Loss of Lysophosphatidylcholine Acyltransferase 1 Leads to Photoreceptor Degeneration in Rd11 Mice

Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice

James S. Friedmana, Bo Changb, Daniel S. Krautha, Irma Lopezc, Naushin H. Waseemd, Ron E. Hurdb, Kecia L. Featherse, Kari E. Branhame, Manessa Shawe, George E. Thomase, Matthew J. Brooksa, Chunqiao Liua, Hirva A. Bakeria, Maria M. Camposf, Cecilia Maubaretd, Andrew R. Websterd, Ignacio R. Rodriguezg, Debra A. Thompsone, Shomi S. Bhattacharyad, Robert K. Koenekoopc, John R. Heckenlivelye, and Anand Swaroopa,e,1

aNeurobiology-Neurodegeneration and Repair Laboratory, fBiological Imaging Core, and gMechanisms of Retinal Diseases Section, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892; bThe Jackson Laboratory, Bar Harbor, ME 04609; cMcGill Ocular Genetics Laboratory, Montreal Children’s Hospital, McGill University Health Centre, Montreal, Quebec H3H-1P3, Canada; dDepartment of Genetics, University College London Institute of Ophthalmology, London EC1V 9EL, United Kingdom; and eDepartment of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105

Edited by Jeremy Nathans, Johns Hopkins University, Baltimore, MD, and approved July 26, 2010 (received for review March 9, 2010)

Retinal degenerative diseases, such as retinitis pigmentosa and (11–13). The study of proteins encoded by these genes is leading Leber congenital amaurosis, are a leading cause of untreatable to a better understanding of the visual transduction pathway, blindness with substantive impact on the quality of life of affected transcription, and ciliogenesis, respectively. individuals and their families. Mouse mutants with retinal dystro- In our ongoing effort to uncover genes critical for retinal func- rd11 JR2845 phies have provided a valuable resource to discover human disease tion or disease, we examined the and B6- strains of genes and helped uncover pathways critical for photoreceptor func- mice that were reported to exhibit retinal degeneration (4). Here tion. Here we show that the rd11 mouse mutant and its allelic strain, we describe the detailed retinal phenotype of these mouse mutants, followed by mapping of the disease locus, and identiﬁ- B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by de- cation of mutations in the Lpcat1 gene that encodes a phospholipid generation of both rods and cones. Using linkage analysis, we map- remodeling enzyme, lysophosphatidylcholine (LPC) acyltransfer- ped the rd11 locus to mouse chromosome 13. We then identiﬁed

Lpcat1 GENETICS – Lpcat1 ase (AT). To assess the physiological impact of mutations, a one-nucleotide insertion (c.420 421insG) in exon 3 of the we have examined membrane association of retinal pigment epi- JR2845 gene. Subsequent screening of this gene in the B6- strain thelium 65-kDa protein (RPE65), measured retinyl ester content, revealed a seven-nucleotide deletion (c.14–20delGCCGCGG) in exon and evaluated dipalmitoylphosphatidylcholine (DPPC), the down- 1. Both sequence changes are predicted to result in a frame-shift, stream product of LPCAT1 enzyme, in rd11 and B6-JR2845 ret- leading to premature truncation of the lysophosphatidylcholine ina. We also report the screening of LPCAT1 in patients with acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is human retinopathy. a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoyl- Results phosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 Retinal Phenotype of the rd11 and B6-JR2845 Mutant Mice. The rd11 and B6-JR2845 mice showed substantially reduced DPPC levels com- and B6-JR2845 strains of mice were identified at The Jackson pared with C57BL/6J control mice, suggesting a causal link to photo- Laboratory in a screen for naturally occurring mouse models of receptor dysfunction. A follow-up screening of LPCAT1 in retinitis retinal disease (4). The two strains are genetically allelic by com- pigmentosa and Leber congenital amaurosis patients did not reveal plementation analysis and do not show any other obvious mor- any obvious disease-causing mutations. Previously, LPCAT1 has been phological or functional phenotype. Retinal fundus examination suggested to be critical for the production of lung surfactant phos- revealed a similar phenotype between both strains. Retinal vessels fl became attenuated by postnatal (P) day 36 with retinal de- pholipids and biosynthesis of platelet-activating factor in nonin am- A matory remodeling pathway. Our studies add another dimension to generation obvious by P113 (Fig. 1 ). Electroretinograms (ERGs) of rd11 mice presented with a reduced rod photoreceptor (dark- an essential role for LPCAT1 in retinal photoreceptor homeostasis. adapted) a-wave, which flattened out at 4 wk of age. Cone photoreceptor (light-adapted) response was similar to WT at 3 wk, but gene discovery | lipid enzyme | phospholipid remodeling | retinal was reduced substantially at 4 and 5 wk. These results suggest that degeneration | visual dysfunction rod photoreceptors are affected before cones (Fig. 1B), consistent with a retinitis pigmentosa-like phenotype. A similar fundus and etinitis pigmentosa (RP) constitutes a group of common ERG profile was observed with B6-JR2845 mice (Fig. S1). Rinherited retinal dystrophies with clinical manifestations, in- Histologically, photoreceptors appeared to develop normally cluding nyctalopia (night blindness) and loss of peripheral vision. in both strains of mice. At P21, no substantial degeneration was One of the early hallmarks of RP is photoreceptor dysfunction that observed in rd11 mice. However, rapid photoreceptor de- is followed by the death of rod and then cone photoreceptors. To generation was seen after P21. By P31, only four to five rows of date, over 40 genes have been associated with inherited forms of RP photoreceptor nuclei were present instead of the usual 10 to 12 (Retnet: www.sph.uth.tmc.edu/retnet/); these genes exhibit distinct patterns of expression (e.g., rod-specific or widely expressed) and encode proteins of diverse biological functions (1–3). Author contributions: J.S.F., B.C., D.S.K., D.A.T., S.S.B., R.K.K., J.R.H., and A.S. designed Animal models of retinal degeneration are a valuable resource research; J.S.F., B.C., D.S.K., I.L., N.H.W., R.E.H., K.L.F., M.S., G.E.T., M.J.B., C.L., H.A.B., in elucidation of genes for human retinal diseases, including RP M.M.C., C.M., and I.R.R. performed research; K.E.B., A.R.W., I.R.R., D.A.T., S.S.B., R.K.K., and Leber congenital amaurosis (LCA) (4, 5). One of the first and J.R.H. contributed new reagents/analytic tools; J.S.F., B.C., I.L., N.H.W., R.E.H., K.L.F., mouse retinal degeneration lines examined, rd1, was defective in M.S., G.E.T., M.J.B., C.L., C.M., I.R.R., D.A.T., S.S.B., R.K.K., and A.S. analyzed data; and J.S.F. Pde6b β and A.S. wrote the paper. , encoding -subunit of cGMP phosphodiesterase and was fl determined to have both an insertion and a point mutation (6, 7). The authors declare no con ict of interest. The rd7 mouse line resulted from the loss of function of orphan This article is a PNAS Direct Submission. nuclear receptor NR2E3 (8, 9), and rd16 mice carried an in-frame Freely available online through the PNAS open access option. deletion in the broadly expressed Cep290 (10). Cross-species 1To whom correspondence should be addressed. E-mail: [email protected]. mutation discovery rapidly occurred in each case once the initial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. disease causing change (in either mice or humans) was identified 1073/pnas.1002897107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1002897107 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Photoreceptor degeneration and loss of ERG signal was observed in rd11 and B6-JR2845 mice. (A) Fundus image of rd11 mice at 24, 36, 113, and 180 d postnatal. Attenuated blood vessels were observed by P36. Large white patches of degeneration are seen by P133. A similar finding was observed in B6- Fig. 2. Photoreceptor disk formation is present in rd11 and B6-JR2845 mice. JR2845 mice (Fig. S1). (B) ERG examination of C57BL/6J (B6) WT at 4 wk of age Retinas from C57BL/6J (B6), rd11, and B6-JR2845 mice were examined by and rd11 mice at 3, 4, and 5 wk. Dark-adapted ERG showed a reduced a-wave in TEM. (A) Nuclei and rod photoreceptor structure appeared to be less densely rd11 mice at 3 wk of age, with a flattening of signal by 4 wk. Light-adapted ERG arranged in mutant mice than in WT (B6). (B) Photoreceptor outer segments was closer to WT at 3 wk of age but showed a substantial reduction of intensity were made in both rd11 and B6-JR2845 mice, suggesting that the mutation at 4 and 5 wk of age. B6-JR2845 mice revealed a similar pattern (Fig. S1). (C) observed did not have an effect on the generation of this structure. H&E staining of rd11,B6-JR2845, and B6 retinas. A substantial loss of photoreceptor nuclei was observed by P31, with a loss to one row of nuclei at P47 in (heart, brain, liver, skeletal muscle, kidney, and pancreas) with rd11 mice. B6-JR2845 photoreceptor nuclei were also obviously reduced by P31, maximum expression in placenta and lung (Fig. S3). Lpcat1 tran- although more nuclei were present at P47 than in rd11 mice. scripts were detectable by RT-PCR in GFP-tagged flow-sorted rod photoreceptors and in mouse retina from embryonic day 12 rows. At P47, only a single row of nuclei remained (Fig. 1C). B6- through 4 mo. (Fig. S3). Quantitative RT-PCR (qRT-PCR) of JR2845 mice demonstrated a similar pattern of degeneration; laser capture microdissected nuclei from retinal outer, inner, and however, more than one row of nuclei was still present by P47 ganglion cell layers suggested broad expression of Lpcat1 in mouse (Fig. 1C). Transmission electron microscopy (TEM) revealed retina (Fig. 4A). outer segments in retinal photoreceptors of both strains (Fig. 2). However, the photoreceptor nuclei of rd11 and B6-JR2845 did Effect of Mutations on Lpcat1 RNA and Protein. Premature stop not appear to be as ordered as B6 control mice. B6-JR2845 codon in transcripts can lead to nonsense-mediated decay (17). retinas showed thinning of the outer nuclear layer at P26, con- RT-PCR analysis showed a 3-fold decrease in the expression of firming a rapid degeneration in these mice (Fig. 2). Lpcat1 in rd11 retina compared with B6 control mice (Fig. 4B), consistent with nonsense-mediated decay. On the other hand, Linkage Analysis and Candidate Gene Screen. To localize the genetic a 2.5-fold increase was detected in retinal Lpcat1 transcripts from defect, we performed linkage analysis of rd11 mice and identified B6-JR2845 mice. Interestingly, the mutation in B6-JR2845 mice is a locus on mouse chromosome 13 between markers D13Mit99 and within a GC-rich region, and a deletion can therefore enhance D13Mit284 (Fig. 3A). This critical genomic region corresponds to Lpcat1 transcription by altering secondary structure. 3.7 Mb with 62 potential candidate genes (Fig. 3B) and is equiv- To examine the effect of mutations on LPCAT1 protein, we alent to human chromosome 7q35 or 5p15. Analysis of additional transfected WT and mutant Lpcat1 cDNA in pcDNA4c vector in mice narrowed the genetic locus to flanking markers D13Mit284 HEK293T cells. Immunoblot analysis of transfected cell extracts and D13Mit255 within an interval of 0.6 Mb harboring 11 genes. using Xpress antibody showed a WT LPCAT1 protein of ∼64 Because of established importance of calcium and high concen- kDa. The rd11 mutation produced a truncated protein around 20 tration of lipids in photoreceptors (14–16), we selected Lpcat1 as kDa, whereas no protein was detected with B6-JR2845 mutation a candidate gene because of its putative Ca2+ binding and AT (Fig. 4C). The anti-LPCAT1 antibody detected endogenous properties. Sequencing of Lpcat1 in rd11 mice revealed a homo- LPCAT1 protein in all lanes. As expected, this protein was smaller zygous one-nucleotide insertion in exon 3 (c.420–421insG) (Fig. than Xpress-tagged LPCAT1 obtained with the WT expression 3D). The WT mouse LPCAT1 protein consists of 534 amino acids; construct (Fig. 4C). The anti-LPCAT1 antibody identifies however, the frame-shift in rd11 mice is predicted to result in an LPCAT1 protein in immunoblots of WT mouse retina but not of abnormal protein after amino acid 140 and a stop after residue rd11 and B6-JR2845 mutants (Fig. 4D). 178. Examination of B6-JR2845 DNA showed a homozygous deletion of seven nucleotides in exon 1 (c.14–20delGCCGCGG) Lipid Analysis. As LPCAT1 enzyme converts LPC to DPPC, we (Fig. 3D), which is predicted to result in a frame-shift after codon predicted that loss of Lpcat1 should affect retinal lipid composi- 8 and a stop codon after 21. These two DNA mutations were not tion. Hence, HPLC and MS analysis was used to profile lipid observed in nine WT mouse strains (Fig. S2). extracts from pooled sets of retina from B6, rd11, and B6-JR2845 mice. HPLC weight-for-weight analysis of lipid headgroups did Lpcat1 Expression Analysis. RNA analysis of a human multiple tis- not reveal a substantive difference between the strains (Fig. 5A). sue Northern blot identified LPCAT1 RNA in all tissues examined Additional experiments showed that within the phosphatidyl-

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1002897107 Friedman et al. Downloaded by guest on September 24, 2021 Lyso-PAF in our analysis. Although lower amounts of PAF are consistent with the loss of LPCAT1 enzyme, it is unclear why PAF and Lyso-PAF are both reduced in amount in mutant mice.

Human Mutation Screen of LPCAT1. Based on the retina-specific phenotype of Lpcat1 mouse mutants, we were encouraged to per- form a mutation screen of exons and boundary regions of LPCAT1 in patients with RP or LCA (Table S2). A multicenter study of 233 patients failed to identify putative disease-causing sequence changes in the LPCAT1 gene. Eight intronic variations, distant from splice acceptor and splice donor sites, were observed. Se- veral silent coding alterations (p.L53L, p.A133A, p.P287P, p.K426K, p.T455T, p.L459L) and common coding alterations (p.L77P, p.T125A, p.M427T) were detected. Five patients with different retinal dystrophy phenotypes showed nonsynonymous amino acid substitutions (p.R114W, p.H266Y, p.A480G, p.A485T, and p.S522L); however, as a second sequence change was not observed in these patients, it is difficult to assign causality or significance. Discussion Lipids have numerous biological roles in membrane bilayers, endocytosis, signaling, and neuroprotection (18–21). Retinal photoreceptors contain a modified primary cilia structure (called the “outer segment”) with highly specialized membrane discs that harbor opsin molecules and other proteins required for phototransduction, the first step in the process of vision. Photoreceptor outer segment discs are apically displaced daily following a circa-

dian rhythm and phagocytosed by the retinal pigment epithelium GENETICS (RPE) (22). The lipid environment of discs plays a critical role in rhodopsin activity and signaling (23, 24). Of the PC in disk lipids, 16:0 (palmitate) is second highest in amount, after 22:6 (doco- Fig. 3. Mutations causing rd11 and B6-JR2845 phenotype are located within sahexaenoic acid, DHA) PC (16). Interestingly, PC and choles- Lpcat1.(A) Haplotypes of rd11 mice. Fifty-six homozygous and 54 heterozy- terol disk lipid composition change over time. Cholesterol and gous rd11 mouse genotypes are shown as black and white blocks, re- fi 16:0 PC are present at high levels in the younger, more basal discs, spectively, over ve markers. Individual mice with recombinations are as and 22:6 PC is greater in older, more apical discs (15, 16). Pio- shown. The initial analysis suggested breakpoints at markers D13Mit284 and neering observations of PC lipid distribution have recently been D13Mit99.(B) Physical map of the genetic region between D13Mit284 and confirmed through newer methods (25). Interestingly, DHA from D13Mit99. Further haplotyping narrowed the genetic interval between plasma is reduced in amount in patients with RP (26) and leads to markers D13Mit284 and D13Mit255. Within this region were 11 candidate transcripts, including Lpcat1.(C) Intron-exon structure for the Lpcat1 gene. reduced retinal function in animals when removed from their diet fi (27). Supplementation with DHA has been observed to be ben- Coding sequences are shown as lled boxes and noncoding regions are un- fi filled. (D) Sequence analysis of Lpcat1 gene in rd11, B6-JR2845 and C57BL/6J e cial in some studies of RP in mouse and man (28, 29). However, (B6) mice. The rd11 strain contained an insertion of a G residue in exon 3. B6- the potentially positive impact of DHA might not be applicable JR2845 mice harbored a seven base-pair deletion in a GC-rich repeat region in across all forms of RP (30). Additionally, 22:6 is a substrate for exon 1. Neither mutation was observed in B6 mice (current figure) or in eight neuroprotectin D1, an antiapoptotic molecule believed to be other control strains (Fig. S2). important for photoreceptor survival (31). Although the importance of DHA in the retina is established, the relationship between loss of DPPC and photoreceptor cell death has not been choline (PC) head group, the ratio of DPPC to the peak lipid class elucidated. Here we report the discovery of Lpcat1 as a retinal (C34:1) was reduced from an average of 84% in B6 mice to 38 to disease gene and show that changes in DPPC levels in the retina 41% in rd11 or B6-JR2845 mice (n = 4 litters for each strain) are associated with photoreceptor dysfunction and degeneration. (Figs. 5 B–E). A concomitant increase in another PC lipid subclass LPCAT1 was recently cloned by two different groups and im- including LPC was not observed (Figs. 5 B–D). These data suggest plicated in several biological functions (32, 33). The LPCAT1 that loss of Lpcat1 has a detrimental effect on DPPC production enzyme, lysophosphatidylcholine acyltransferase 1, preferentially in the retina. transfers palmitoyl-CoA to LPC and is part of the lipid remod- As mutant mice exhibited lower retinal levels of DPPC, we eling pathway (Lands’ cycle) used to generate lipid diversity after hypothesized that adding DPPC to their diet may have a beneficial de novo synthesis (32, 33). Because of its high expression in lung, it effect. Breeding cages were therefore supplemented with 2 g/kg was hypothesized that LPCAT1 generates DPPC, an important DPPC in mouse chow, and the resulting pups were examined. component of lung surfactant (32, 33). A recent knock-in mouse Retinal histology of adult rd11 or B6-JR2845 mice supplemented introducing the β-galactosidase gene after exon 9 of Lpcat1 had with DPPC chow did not demonstrate any significant change from reduced AT activity compared with the WT. The reduced func- mutants fed with unsupplemented chow (Fig. S4). We therefore tion of LPCAT1 with subsequent loss of DPPC was suggested to checked if dietary supplementation of DPPC affects retinal lipid be critical for transitioning to air breathing in mice (34). In our composition. No change in DPPC levels was observed in supple- investigations, we did not observe an obvious pathology in rd11 mented P22 rd11 mouse retina compared with unsupplemented and B6-JR2845 mice consistent with the loss of surfactant; how- rd11 mutants (n =3)(Fig. S5). ever, it is possible that such a phenotype may be induced under As LPCAT1 may have a role in the noninflammatory pathway, appropriate stress conditions. Additional study of rd11 and B6- we decided to measure PAF C16 and lyso-platelet activating JR2845 pups after birth may reveal the phenotype described factor (Lyso-PAF) C16. We observed a lower amount of both above. In vitro experiments suggest the existence of compensatory lipids in mutant mice than WT (n = 3 for each strain) (Table S1). mechanisms in lung epithelial cells when LPCAT1 is overex- Notably, some measurements of Lyso-PAF were below our pressed (35). Whether or not such compensation occurs in rd11 threshold of detection and were calculated as being absent from and B6-JR2845 mice remains to be elucidated. As DPPC is not the respective sample. This result may lead to a lower estimate of completely lost in retinas of rd11 and B6-JR2845 mice, it is pos-

Friedman et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Fig. 4. Expression of Lpcat1 in WT and mutant retina. (A) Quantita- tive RT-PCR analysis was performed with Lpcat1 from laser-capture microdissected cells collected from glangion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Samples were normalized to Hprt. For each gene tested, ΔCt qRT-PCR values were summed and displayed in graphical format. POU class 4 homeobox 2 (Pou4f2), glutamate receptor, metabotropic 6 (Grm6), and Rho- dopsin, (Rho) were used as controls for GCL, INL, and ONL, respectively. Lpcat1 was present in all nuclear layers. Error bars show SD of the mean. (B) Quantitative RT-PCR of retinal RNA from C57BL/ 6J, rd11, and B6-JR2845 mice to test for nonsense-mediated decay. Lpcat1 expression fold changes of −3.1 and 2.5 were observed in rd11 and B6-JR2845 mice, respectively, compared with C57BL/6J control. (C) Western blot of HEK 293T cells transfected with Xpress-tagged WT or mutant Lpcat1 cDNA. UNTR, untransfected. Lpcat1 cDNA containing the rd11 mutation generated a truncated protein product and no protein was observed with the B6-JR2845 mutation. The same immunoblot incubated with anti-LPCAT1 antibody demonstrated an endogenous band at the expected size in all lanes with a higher band corresponding to the Xpress-tagged LPCAT1 protein in the Xpress-RD11 transfected lane. (D) Immunoblot of retinas from C57BL/6J (adult), rd11 (P22), and B6-JR2845 (P21) mice. Anti-LPCAT1 antibody showed cross-reactivity to multiple proteins in addition to one at the expected size (59 kDa). The 59-kDa band was observed in WT mice, but was not present in the rd11 or B6-JR2845 lanes. β-Actin was used as a protein loading control (C and D).

sible that other LPCAT family members are able to partially research is needed to understand the underlying biochemical compensate for the loss of LPCAT1. mechanism of photoreceptor cell death caused by the loss of In addition to its LPCAT function, LPCAT1 also acts as lyso- LPCAT1 function. PAF AT and is thought to have a role in the noninflammatory PAF Because the addition of DHA to diet had a beneficial effect on remodeling pathway (36). LPCAT1 is highly expressed in co- retinal degeneration (28), we performed a similar experiment us- lorectal cancer cells and suggested to influence membrane fluidity, ing DPPC supplementation. In our studies, the supplemented diet potentially playing a role in cancer cells’ progression toward me- did not change retinal DPPC content or improve pathology in rd11 tastasis (37). Recently, another study observed a decrease in or B6-JR2845 mice. We suggest that a larger dietary amount of Lpcat1 expression in retinas of diabetic mice (38). Our data further DPPC, another form of lipid, or a different intake method might emphasize the importance of LPCAT1 in diverse cellular probe needed to rescue the Lpcat1 mutant phenotype. Alternatively, cesses, with an as yet undefined new role in photoreceptor biology. local synthesis of DPPC is required to meet photoreceptor needs. The retinal phenotypes of Lpcat1 mutant mice raise important In conclusion, we have identified a unique gene for retinal dys- mechanistic questions. The presence of outer segment discs in trophy in mice and highlighted the importance of maintaining rd11 and B6-JR2845 retina suggests that LPCAT1 activity and specific lipid profiles in photoreceptors. A few other human retinal appropriate DPPC levels are not essential for photoreceptor dystrophy genes (ELOVL4, ABCA4, REP-1,andProminin)are morphogenesis but may be critical for homeostasis and functional believed to be involved in lipid metabolism, transport, protein lip- maintenance. Consistent with this, a recent qRT-PCR analysis idation, and formation of membrane protrusions (51–56). Although shows that Lpcat1 expression is increased from P2 to P25 (39). we could not establish disease causality in our patient cohort, Further investigations are required to precisely delineate the role LPCAT1 remains a strong candidate gene for human retinal de- of LPCAT1 in the retina and photoreceptors. generation because of the importance of DPPC and lipid metabo- Photoreceptor-RPE interaction is crucial for the retinoid cycle lism in retina. It is possible that LPCAT1 mutations have a more during phototransduction and two critical proteins of this cycle pleiotropic effect in humans. Hence, further LPCAT1 mutation are lecithin retinol AT and RPE65 (40). DPPC is involved in the screening in patients with both syndromic and nonsyndromic retinal formation of retinyl esters through lecithin retinol AT (41, 42). degeneration is desirable. Our studies show that elucidation of DPPC is also thought to act as a palmitoylation switch for RPE65 LPCAT1 function and biology is expected to provide fundamental (43), although this hypothesis is controversial (44). We therefore insights into diverse physiological processes and disease. tested the two hypotheses through the measurement of retinyl esters and by examining putative soluble and membrane-bound Materials and Methods RPE65 isoforms, respectively, in whole eye and RPE of WT and Histological and ERG Analysis of rd11 and B6-JR2845 Mice. Animal work was mutant mice. No significant change in retinyl ester content or performed with approval from the National Eye Institute and The Jackson RPE65 localization was observed (Fig. S6), suggesting that altered Laboratory Animal Care and Use Committees. Histology and ERGs were DPPC levels do not have significant impact on these processes. carried out as previously described (57). LPCAT1 plays a role in the noninflammatory response to oxi- dization and inflammation (38). The loss of LPCAT1 could lead Linkage Analysis of rd11 Mice. Genotyping of mice and linkage analysis of rd11 to increased presence or slower enzymatic processing of LPC that, mice were completed as previously described (57). Genetic markers defining 2+ in turn, can lead to membrane disruption (45) and Ca influx the critical domain were D13Mit284 and D13Mit99. The physical region cor- (46), either of which may then contribute to photoreceptor cell responding these markers outlines a 3.7-Mb region containing 62 genes. Ad- death. As we did not observe an increase in LPC amount, this is ditional screening totaling 1,301 samples narrowed the disease locus to unlikely to be the cause of disease in these mice. LPCAT1 loss markers D14Mit255 and D13Mit284. This genetic area consists of 11 genes, may also lead to slower alkyl-PC formation, part of a non- including Lpcat1. inflammatory PAF inactivation pathway (38). This hypothesis would predict that increased levels of lyso-PAF would be gener- Mouse Mutation Screen. We examined BC005662/Aytl2/Lpcat1 as a candidate ated, allowing for a greater inflammatory response after light gene for the rd11 and B6-JR2845 mice. Lpcat1 was selected because of its damage. Inflammation and immune response play critical roles in putative Ca+ binding domain and AT activity. Primers for all 14 exons are etiology of age-related macular degeneration (47–49) and reti- included in Table S3. Mouse strains A/J, AE/J, BALB/cJ, C3H/cJ, C57BL/6J, CAST1/ nopathies (50). LPCAT1’s role in the noninflammatory pathway EiJ, DBA/2J, MOLC/Rk, and NON/Lt were used as controls. would lead to a decrease in PAF and an increase in Lyso-PAF (36); however, lower amounts of PAF and Lyso-PAF were ob- Transmission Electron Microscopy. C57BL/6J (B6), rd11 and B6-JR2845 mouse served in both mutant strains compared with WT mice. Further retinas at ages P27, P21, and P26, respectively, were prepared similar to that

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1002897107 Friedman et al. Downloaded by guest on September 24, 2021 previously described (58). Samples were analyzed through the University of Michigan Electron Microscopy core.

Laser Capture Microdissection. An enucleated mouse eye was ﬂash-frozen in 100% OCT. Then, 10-μm thick sections were placed on PALM Membrane slides nuclease-free (Zeiss), dehydrated in 70% alcohol for 2 min, and stained with Cresyl Violet staining (LCM Staining Kit, Ambion) for 30 s and dehydrated in 70 and 100% alcohol for visualization of cell nuclei. The sections were microdissected using the laser capture method [PALM Microlaser System; Laser Microdissecton and Pressure Catapulting (LMPC), Zeiss]. The cells were collected from the GCL, INL, and ONL. Cells were stored at −80 °C.

RNA-Based Analyses. Laser-capture microdissected cellular RNA was extracted through standard methods (Qiagen), reverse-transcribed, and amplified using WT-Ovation One-Direct kit (Nugen). TaqMan Gene Expression Assays (Applied Biosystems) using Lpcat1 (Mm00461015_m1) were carried out on each amplified cDNA according to the manufacturer’s protocol. POU class 4 homeobox 2 (Pou4f2, Mm00454754_s1), glutamate receptor metabotropic 6 (Grm6, Mm00841148_m1), and Rhodopsin, (Rho, Mm00520345_m1) were used as controls for the GCL, INL, and ONL, respectively. Samples were normalized to Hprt (Mm01310747_g1). PCR amplifications were performed in triplicate. RT- PCR and Northern blot analysis on WT mouse retinal RNA and on a human multiple tissue Northern blot (Clontech) were carried out as previously described (59). Primers used for RT-PCR are listed in Table S3. For the nonsense- mediated decay test, qRT-PCR was performed in triplicate on three sets of P21- to P23-aged retina cDNA samples derived from each mouse strain (B6, rd11, B6-JR2845). Lpcat1 and Hprt TaqMan Gene Expression Assays were used −ΔΔCt as described above. The comparative CT, or 2 , method of analysis was performed using Hprt for normalization of expression levels. GENETICS Immunoblot Analysis. Lpcat1 cDNA was amplified and cloned into the pcDNA4c vector (Invitrogen) and the rd11 and B6-JR2845 mutations were introduced through site-directed mutagenesis. Primers used for cloning and mutagenesis reactions are listed in Table S3. HEK293T cell transfection, harvesting and im- munoblotting were performed as previously described (59). Anti-Xpress monoclonal antibody (1:4,000; Invitrogen, 46–0528), anti–β-actin (1:5,000; Sigma-Aldrich, A2228) primary antibody and anti-AYTL2 (LPCAT1) antibody (1:750; Sigma-Aldrich, HPA022268) were used on the HEK293T immunoblot. Retinas from adult B6, P22 rd11 and P21 B6-JR2845 mice were sonicated directly in 2× SDS buffer with protease inhibitor, and run out by SDS/PAGE. Anti-LPCAT1 antibody (1:750) was preadsorbed on an immunoblot with retinal extracts from P22 rd11, and P21 B6-JR2845 mice before being used experimentally. Mouse anti-rabbit HRP conjugated light chain specific antibody (1:4,000; Millipore, MAB201P) was used to detect the primary antibody. Mouse anti–β-actin antibody was used as a control for loading.

Retinal Lipid Extractions, HPLC, and MS Analysis. Retinas from B6, rd11, and B6- JR2845 mice were collected at P21 to P24 and lipids were extracted using the Bligh and Dyer protocol (60). One litter of pups was pooled for each experiment and three sets of lipids were examined for each strain. Quantitative phospholipid analysis (weight % analysis) was performed by HPLC by Avanti Polar Lipids. Semiquantitative analysis of major phospholipid species although flow-infusion MS was also carried out by Avanti Polar Lipids. PAFs Fig. 5. Mutations in Lpcat1 result in reduced retinal levels of DPPC. (A)HPLC were separated in a Waters Corp. Symmetry 300 C-18 column (3.5 μm, 2.1 × analysis showing percent weight for weight. Retinal lipids from four litters of 150 mm) using an Agilent Technologies Series 1200 HPLC equipped with each strain were extracted and subjected to HPLC analysis. Averages of CH, Cer, a binary pump and automatic sample injector. The column was run at 0.1 mL/ PE, PI, and PC lipid groups are shown as a percent weight for weight (wt/wt) min and the PAFs separated using a gradient starting with 25% solvent A with SE. No substantive difference in the PC lipid group was observed among (20% acetonitrile, 80% water 5 mM ammonium acetate) and 75% solvent B C57BL/6J (B6), B6-JR2845 or rd11 mice. CH, cholesterol; Cer, ceramide, PE, (84% methanol, 16% methylene chloride, 1 mM ammonium acetate). The phosphatidylethanolamine, PI, phosphatidylinositol, PC, phosphstidylcholine. gradient increased linearly to 100% solvent B in 20 min and was maintained at (B–D) Retinal lipid extracts from B6 (B), rd11 (C), and B6-JR2845 (D) mice were 100% B until 50 min. The solvents were then changed to original conditions examined by HPLC and MS. Within the phosphatidylcholine headgroup class, (25% B) for an additional 10 min to reequilibrate the column. The total run the m/z of 735.1, corresponding to DPPC, was substantially reduced relative to time was 60 min. Quantification of PAFs was performed by MS using a Waters/ the peak lipid m/z observed in both rd11 and B6-JR2845. The observed loss was Micro mass QTOF micro equipped with an electron spray probe (ESi). The significant when compared with B6 (t test, P < 0.001). (E)DPPCcontent entire flow of the LC was diverted into the QTOF. The ESi probe settings were expressed as a percentage (%) of peak lipid. PC lipids from retinal extracts were as follows: capillary voltage, 3,000 v; sample cone, 35; extraction cone, 3.0; subjected to MS/MS analysis. The peak corresponding to DPPC was compared source temp, 120 °C; desolation temp, 180 °C. with the peak lipid class in the PC headgroup and expressed as a percentage of peak. The data presented is an average of four sets of extracts from each strain Human Mutation Screen. We examined retinal dystrophy patients from the with SE shown. A student’s t test showed a significant difference between B6 United States, Canada, and the United Kingdom. Human mutation screens were − − and B6-JR2845 (P = 7.63 × 10 5), and between B6 and rd11 (P = 5.54 × 10 4), but approved by the University of Michigan Institutional Review Board, McGill not between B6-JR2845 and rd11 (P = 0.223). Research Ethics Board at Montreal Children’s Hospital, and Moorfields Eye Hospital Ethics Committee, respectively. Primers designed for this study are listed in Table S3. For the Michigan and United Kingdom study, LPCAT1 exon PCR products were amplified and sequenced through standard methods. The Cana-

Friedman et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 dian/McGill study tested a total of 50 patients (25 patients with LCA and 25 with ACKNOWLEDGMENTS. We thank Jessica Chang, Mohammad Othman, and autosomal recessive juvenile-onset RP). Patients were strongly selected as the Renee Pigeon for assistance and the National Eye Institute Biological Imag- then known seven LCA genes (GUCY2D, RPE65, AIPL1, CRX, CRB1, RPGRIP1, ing Core for equipment usage. Funding for this research was provided by the RDH12) were excluded by sequencing and all patients were consanguineous to National Eye Institute Intramural program, National Institutes of Health improve chances of finding mutations. PCR products of each exon (including Grant EY019943, Foundation Fighting Blindness, Research to Prevent Blind- flanking introns) were mixed with equal amounts of normal control and ex- ness, Canadian Institutes of Health Research, Foundation Fighting Blindness- amined by DHPLC. Trace profiles were examined by visual inspection of chro- Canada, Fonds de la Recherche en Santé Québec, Reseau de Vision, and matograms by two independent observers and compared with the WT DNA Foundation for Retinal Research. G.E.T. was a recipient of a Fight for Sight fragment profile. Samples with aberrant profiles were subsequently sequenced. Summer Student Fellowship.

1. Hartong DT, Berson EL, Dryja TP (2006) Retinitis pigmentosa. Lancet 368:1795–1809. 31. Bazan NG (2007) Homeostatic regulation of photoreceptor cell integrity: Significance 2. Trifunovic D, et al. (2008) A high-resolution RNA expression atlas of retinitis pigmentosa of the potent mediator neuroprotectin D1 biosynthesized from docosahexaenoic acid: genes in human and mouse retinas. Invest Ophthalmol Vis Sci 49:2330–2336. The Proctor Lecture. Invest Ophthalmol Vis Sci 48:4866–4881; biography 4864–4865. 3. Daiger SP, Bowne SJ, Sullivan LS (2007) Perspective on genes and mutations causing 32. Chen X, Hyatt BA, Mucenski ML, Mason RJ, Shannon JM (2006) Identification and retinitis pigmentosa. Arch Ophthalmol 125(2):151–158. characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells. 4. Chang B, et al. (2005) Mouse models of ocular diseases. Vis Neurosci 22:587–593. Proc Natl Acad Sci USA 103:11724–11729. 5. Baehr W, Frederick JM (2009) Naturally occurring animal models with outer retina 33. Nakanishi H, et al. (2006) Cloning and characterization of mouse lung-type acyl-CoA: phenotypes. Vision Res 49:2636–2652. lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells 6. Bowes C, et al. (1990) Retinal degeneration in the rd mouse is caused by a defect in and possible involvement in surfactant production. J Biol Chem 281:20140–20147. the beta subunit of rod cGMP-phosphodiesterase. Nature 347:677–680. 34. Bridges JP, et al. (2010) LPCAT1 regulates surfactant phospholipid synthesis and is 7. Pittler SJ, Baehr W (1991) Identification of a nonsense mutation in the rod required for transitioning to air breathing in mice. J Clin Invest 120:1736–1748. photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse. Proc 35. Butler PL, Mallampalli RK (2009) Cross-talk between remodeling and de novo pathways Natl Acad Sci USA 88:8322–8326. maintains phospholipid balance through ubiquitination. J Biol Chem 285:6246–6258. 8. Akhmedov NB, et al. (2000) A deletion in a photoreceptor-specific nuclear receptor 36. Harayama T, Shindou H, Ogasawara R, Suwabe A, Shimizu T (2008) Identification of mRNA causes retinal degeneration in the rd7 mouse. Proc Natl Acad Sci USA 97: a novel noninflammatory biosynthetic pathway of platelet-activating factor. J Biol 5551–5556. Chem 283:11097–11106. 9. Chen J, Rattner A, Nathans J (2006) Effects of L1 retrotransposon insertion on 37. Mansilla F, et al. (2009) Lysophosphatidylcholine acyltransferase 1 (LPCAT1) over- transcript processing, localization and accumulation: Lessons from the retinal expression in human colorectal cancer. JMolMed87(1):85–97. degeneration 7 mouse and implications for the genomic ecology of L1 elements. Hum 38. Cheng L, Han X, Shi Y (2009) A regulatory role of LPCAT1 in the synthesis of Mol Genet 15:2146–2156. inflammatory lipids, PAF and LPC, in the retina of diabetic mice. Am J Physiol 10. Chang B, et al. (2006) In-frame deletion in a novel centrosomal/ciliary protein CEP290/ Endocrinol Metab 297:E1276–E1282. NPHP6 perturbs its interaction with RPGR and results in early-onset retinal 39. Tummala P, Mali RS, Guzman E, Zhang X, Mitton KP (2010) Temporal ChIP-on-Chip of degeneration in the rd16 mouse. Hum Mol Genet 15:1847–1857. RNA-Polymerase-II to detect novel gene activation events during photoreceptor 11. McLaughlin ME, Sandberg MA, Berson EL, Dryja TP (1993) Recessive mutations in the maturation. Mol Vis 16:252–271. gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis 40. Travis GH, Golczak M, Moise AR, Palczewski K (2007) Diseases caused by defects in the pigmentosa. Nat Genet 4(2):130–134. visual cycle: Retinoids as potential therapeutic agents. Annu Rev Pharmacol Toxicol 12. den Hollander AI, et al. (2006) Mutations in the CEP290 (NPHP6) gene are a frequent 47:469–512. cause of Leber congenital amaurosis. Am J Hum Genet 79:556–561. 41. Barry RJ, Cañada FJ, Rando RR (1989) Solubilization and partial purification of retinyl 13. Haider NB, et al. (2000) Mutation of a nuclear receptor gene, NR2E3, causes enhanced ester synthetase and retinoid isomerase from bovine ocular pigment epithelium. J S cone syndrome, a disorder of retinal cell fate. Nat Genet 24(2):127–131. Biol Chem 264:9231–9238. 14. Sancho-Pelluz J, et al. (2008) Photoreceptor cell death mechanisms in inherited retinal 42. Saari JC, Bredberg DL (1989) Lecithin:retinol acyltransferase in retinal pigment degeneration. Mol Neurobiol 38:253–269. epithelial microsomes. J Biol Chem 264:8636–8640. 15. Boesze-Battaglia K, Fliesler SJ, Albert AD (1990) Relationship of cholesterol content to 43. Xue L, Gollapalli DR, Maiti P, Jahng WJ, Rando RR (2004) A palmitoylation switch spatial distribution and age of disc membranes in retinal rod outer segments. J Biol mechanism in the regulation of the visual cycle. Cell 117:761–771. Chem 265:18867–18870. 44. Jin M, Yuan Q, Li S, Travis GH (2007) Role of LRAT on the retinoid isomerase activity 16. Albert AD, Young JE, Paw Z (1998) Phospholipid fatty acyl spatial distribution in and membrane association of Rpe65. J Biol Chem 282:20915–20924. bovine rod outer segment disk membranes. Biochim Biophys Acta 1368(1):52–60. 45. Weltzien HU (1979) Cytolytic and membrane-perturbing properties of lysophos- 17. Nicholson P, et al. (2010) Nonsense-mediated mRNA decay in human cells: phatidylcholine. Biochim Biophys Acta 559:259–287. Mechanistic insights, functions beyond quality control and the double-life of NMD 46. Wilson-Ashworth HA, et al. (2004) Formation of transient non-protein calcium pores factors. Cell Mol Life Sci 67:677–700. by lysophospholipids in S49 Lymphoma cells. J Membr Biol 200(1):25–33. 18. van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: Where they are and 47. Gehrs KM, Anderson DH, Johnson LV, Hageman GS (2006) Age-related macular how they behave. Nat Rev Mol Cell Biol 9(2):112–124. degeneration—Emerging pathogenetic and therapeutic concepts. Ann Med 38:450–471. 19. Wymann MP, Schneiter R (2008) Lipid signalling in disease. Nat Rev Mol Cell Biol 9(2): 48. Nussenblatt RB, Liu B, Li Z (2009) Age-related macular degeneration: An 162–176. immunologically driven disease. Curr Opin Investig Drugs 10:434–442. 20. Piomelli D, Astarita G, Rapaka R (2007) A neuroscientist’s guide to lipidomics. Nat Rev 49. Swaroop A, Chew EY, Rickman CB, Abecasis GR (2009) Unraveling a multifactorial Neurosci 8:743–754. late-onset disease: From genetic susceptibility to disease mechanisms for age-related 21. Bazan NG (2006) Cell survival matters: Docosahexaenoic acid signaling, neuroprotection macular degeneration. Annu Rev Genomics Hum Genet 10:19–43. and photoreceptors. Trends Neurosci 29:263–271. 50. Heckenlively JR, Ferreyra HA (2008) Autoimmune retinopathy: A review and 22. LaVail MM (1976) Rod outer segment disk shedding in rat retina: Relationship to cyclic summary. Semin Immunopathol 30(2):127–134. lighting. Science 194:1071–1074. 51. Zhang K, et al. (2001) A 5-bp deletion in ELOVL4 is associated with two related forms 23. Watts A, Davoust J, Marsh D, Devaux PF (1981) Distinct states of lipid mobility in of autosomal dominant macular dystrophy. Nat Genet 27(1):89–93. bovine rod outer segment membranes. Resolution of spin label results. Biochim 52. Agbaga MP, et al. (2008) Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the Biophys Acta 643:673–676. biosynthesis of very long chain fatty acids. Proc Natl Acad Sci USA 105:12843–12848. 24. Brown MF (1994) Modulation of rhodopsin function by properties of the membrane 53. Allikmets R, et al. (1997) A photoreceptor cell-specific ATP-binding transporter gene bilayer. Chem Phys Lipids 73(1–2):159–180. (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet 15:236–246. 25. Hayasaka T, et al. (2008) Matrix-assisted laser desorption/ionization quadrupole ion 54. Molday RS, Zhong M, Quazi F (2009) The role of the photoreceptor ABC transporter trap time-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry reveals ABCA4 in lipid transport and Stargardt macular degeneration. Biochim Biophys Acta a layered distribution of phospholipid molecular species in the mouse retina. Rapid 1791:573–583. Commun Mass Spectrom 22:3415–3426. 55. Seabra MC, Ho YK, Anant JS (1995) Deficient geranylgeranylation of Ram/Rab27 in 26. Gong J, et al. (1992) Plasma docosahexaenoic acid levels in various genetic forms of choroideremia. J Biol Chem 270:24420–24427. retinitis pigmentosa. Invest Ophthalmol Vis Sci 33:2596–2602. 56. Maw MA, et al. (2000) A frameshift mutation in prominin (mouse)-like 1 causes 27. Leat WM, Curtis R, Millichamp NJ, Cox RW (1986) Retinal function in rats and guinea- human retinal degeneration. Hum Mol Genet 9(1):27–34. pigs reared on diets low in essential fatty acids and supplemented with linoleic or 57. Pang JJ, et al. (2005) Retinal degeneration 12 (rd12): A new, spontaneously arising linolenic acids. Ann Nutr Metab 30(3):166–174. mouse model for human Leber congenital amaurosis (LCA). Mol Vis 11:152–162. 28. Ebert S, et al. (2009) Docosahexaenoic acid attenuates microglial activation and 58. Bechtold LS, Smith RS (2002) Systematic evaluation of the mouse eye, ed Smith RS delays early retinal degeneration. J Neurochem 110:1863–1875. (CRC Press, Boca Raton), pp 272–277. 29. Hoffman DR, et al. (2004) A randomized, placebo-controlled clinical trial of 59. Friedman JS, et al. (2006) Premature truncation of a novel protein, RD3, exhibiting docosahexaenoic acid supplementation for X-linked retinitis pigmentosa. Am J subnuclear localization is associated with retinal degeneration. Am J Hum Genet 79: Ophthalmol 137:704–718. 1059–1070. 30. Li F, et al. (2009) DHA does not protect ELOVL4 transgenic mice from retinal 60. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. degeneration. Mol Vis 15:1185–1193. Can J Biochem Physiol 37:911–917.

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