Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease
2016; Elsevier BV; Volume: 186; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2016.03.013
ISSN1525-2191
AutoresScott H. Greenwald, Jeremy R. Charette, Magdalena Staniszewska, Lan Ying Shi, Steve D. M. Brown, Lisa Stone, Qin Liu, Wanda L. Hicks, Gayle B. Collin, Michael R. Bowl, Mark P. Krebs, Patsy M. Nishina, Eric A. Pierce,
Tópico(s)Advanced Fluorescence Microscopy Techniques
ResumoThe nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD+ in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea–generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1V9M mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD+ metabolism. The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD+ in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea–generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1V9M mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD+ metabolism. Leber congenital amaurosis (LCA), a genetically heterogeneous retinal degenerative disease associated with mutations in at least 18 genes, represents the most common cause (10% to 18%) of incurable blindness in children1Perrault I. Hanein S. Zanlonghi X. Serre V. Nicouleau M. Defoort-Delhemmes S. Delphin N. Fares-Taie L. Gerber S. Xerri O. Edelson C. Goldenberg A. Duncombe A. Le Meur G. Hamel C. Silva E. Nitschke P. Calvas P. Munnich A. Roche O. Dollfus H. Kaplan J. Rozet J.M. Mutations in NMNAT1 cause Leber congenital amaurosis with early-onset severe macular and optic atrophy.Nat Genet. 2012; 44: 975-977Crossref PubMed Scopus (100) Google Scholar, 2den Hollander A.I. Roepman R. Koenekoop R.K. Cremers F.P. Leber congenital amaurosis: genes, proteins and disease mechanisms.Prog Retin Eye Res. 2008; 27: 391-419Crossref PubMed Scopus (616) Google Scholar and accounts for ≥5% of all inherited retinal degenerations.3Koenekoop R.K. An overview of Leber congenital amaurosis: a model to understand human retinal development.Surv Ophthalmol. 2004; 49: 379-398Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Across all forms of LCA, vision is impaired at birth or begins deteriorating during early infancy because of retinal degeneration.4den Hollander A.I. Black A. Bennett J. Cremers F.P. 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Consugar M. Webster A.R. Sahel J.A. Moore A.T. Berson E.L. Liu Q. Gai X. Pierce E.A. NMNAT1 mutations cause Leber congenital amaurosis.Nat Genet. 2012; 44: 1040-1045Crossref PubMed Scopus (141) Google Scholar of LCA cases are caused by mutations in the NMNAT1 gene,1Perrault I. Hanein S. Zanlonghi X. Serre V. Nicouleau M. Defoort-Delhemmes S. Delphin N. Fares-Taie L. Gerber S. Xerri O. Edelson C. Goldenberg A. Duncombe A. Le Meur G. Hamel C. Silva E. Nitschke P. Calvas P. Munnich A. Roche O. Dollfus H. Kaplan J. Rozet J.M. Mutations in NMNAT1 cause Leber congenital amaurosis with early-onset severe macular and optic atrophy.Nat Genet. 2012; 44: 975-977Crossref PubMed Scopus (100) Google Scholar, 8Falk M.J. Zhang Q. Nakamaru-Ogiso E. Kannabiran C. Fonseca-Kelly Z. Chakarova C. Audo I. Mackay D.S. Zeitz C. Borman A.D. Staniszewska M. Shukla R. Palavalli L. Mohand-Said S. Waseem N.H. Jalali S. Perin J.C. Place E. Ostrovsky J. Xiao R. Bhattacharya S.S. Consugar M. Webster A.R. Sahel J.A. 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Ziegler M. The NAD metabolome–a key determinant of cancer cell biology.Nat Rev Cancer. 2012; 12: 741-752Crossref PubMed Scopus (419) Google Scholar In the Golgi complex and mitochondria, two other NMNAT isoforms that similarly serve to regenerate NAD+ are encoded by NMNAT2 and NMNAT3, respectively.17Lau C. Niere M. Ziegler M. The NMN/NaMN adenylyltransferase (NMNAT) protein family.Front Biosci (Landmark Ed). 2009; 14: 410-431Crossref PubMed Scopus (95) Google Scholar, 19Berger F. Lau C. Dahlmann M. Ziegler M. Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms.J Biol Chem. 2005; 280: 36334-36341Crossref PubMed Scopus (378) Google Scholar However, neither NMNAT2 nor NMNAT3 compensates for the loss of NMNAT1 function, considering that homozygous knockout of Nmnat1 in mice results in embryonic lethality.20Conforti L. Janeckova L. Wagner D. Mazzola F. Cialabrini L. Di Stefano M. Orsomando G. 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Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.Nat Neurosci. 2001; 4: 1199-1206Crossref PubMed Scopus (502) Google Scholar Given that NMNAT1 is expressed ubiquitously,9Chiang P.W. Wang J. Chen Y. Fu Q. Zhong J. Chen Y. Yi X. Wu R. Gan H. Shi Y. Chen Y. Barnett C. Wheaton D. Day M. Sutherland J. Heon E. Weleber R.G. Gabriel L.A. Cong P. Chuang K. Ye S. Sallum J.M. Qi M. Exome sequencing identifies NMNAT1 mutations as a cause of Leber congenital amaurosis.Nat Genet. 2012; 44: 972-974Crossref PubMed Scopus (102) Google Scholar, 19Berger F. Lau C. Dahlmann M. Ziegler M. Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms.J Biol Chem. 2005; 280: 36334-36341Crossref PubMed Scopus (378) Google Scholar it is unclear why abnormal NMNAT1 function causes a retina-specific disease. The precise mechanism(s) underlying retinal cell degeneration in NMNAT1-LCA also has not been defined, and no treatment is available for patients. Thus far, in the absence of an animal model, mutant NMNAT1 has been studied in human embryonic kidney cells (HEK293T), human cervical cancer cells (HeLa), mouse dorsal root ganglia, NMNAT1-LCA patient fibroblasts, and NMNAT1-LCA patient red blood cells (nonnucleated),8Falk M.J. Zhang Q. Nakamaru-Ogiso E. Kannabiran C. Fonseca-Kelly Z. Chakarova C. Audo I. Mackay D.S. Zeitz C. Borman A.D. Staniszewska M. Shukla R. Palavalli L. Mohand-Said S. Waseem N.H. Jalali S. Perin J.C. Place E. Ostrovsky J. Xiao R. Bhattacharya S.S. Consugar M. Webster A.R. Sahel J.A. Moore A.T. Berson E.L. Liu Q. Gai X. Pierce E.A. NMNAT1 mutations cause Leber congenital amaurosis.Nat Genet. 2012; 44: 1040-1045Crossref PubMed Scopus (141) Google Scholar, 10Koenekoop R.K. Wang H. Majewski J. Wang X. Lopez I. Ren H. Chen Y. Li Y. Fishman G.A. Genead M. Schwartzentruber J. Solanki N. Traboulsi E.I. Cheng J. Logan C.V. McKibbin M. Hayward B.E. Parry D.A. Johnson C.A. Nageeb M. Poulter J.A. Mohamed M.D. Jafri H. Rashid Y. Taylor G.R. Keser V. Mardon G. Xu H. Inglehearn C.F. Fu Q. Toomes C. Chen R. Finding of Rare Disease Genes (FORGE) Canada ConsortiumMutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration.Nat Genet. 2012; 44: 1035-1039Crossref PubMed Scopus (142) Google Scholar, 27Sasaki Y. Margolin Z. Borgo B. Havranek J.J. Milbrandt J. Characterization of Leber's congenital amaurosis-associated NMNAT1 mutants.J Biol Chem. 2015; 290: 17228-17238Crossref PubMed Scopus (29) Google Scholar, 28Siemiatkowska A.M. Schuurs-Hoeijmakers J.H. Bosch D.G. Boonstra F.N. Riemslag F.C. Ruiter M. de Vries B.B. den Hollander A.I. Collin R.W. Cremers F.P. Nonpenetrance of the most frequent autosomal recessive leber congenital amaurosis mutation in NMNAT1.JAMA Ophthalmol. 2014; 132: 1002-1004Crossref PubMed Scopus (22) Google Scholar with inconsistent results. Further, in vitro systems do not allow for the assessment of whether a treatment preserves vision and cannot address practical challenges associated with delivering therapeutic agents to the NMNAT1-LCA retina. For instance, determining which specific cell type(s) should be the therapeutic target may be critical for a successful intervention, given that the effects of NMNAT1 overexpression in otherwise healthy retinal cells are unknown. Here, we report the discovery and characterization of two mutant Nmnat1 mouse lines derived from chemical mutagenesis programs.29Coghill E.L. Hugill A. Parkinson N. Davison C. Glenister P. Clements S. Hunter J. Cox R.D. Brown S.D. A gene-driven approach to the identification of ENU mutants in the mouse.Nat Genet. 2002; 30: 255-256Crossref PubMed Scopus (155) Google Scholar, 30Won J. Shi L.Y. Hicks W. Wang J. Naggert J.K. Nishina P.M. Translational vision research models program.Adv Exp Med Biol. 2012; 723: 391-397Crossref PubMed Scopus (11) Google Scholar Wild-type mice were injected with a potent mutagen, N-ethyl-N-nitrosourea (ENU), which introduces point mutations in DNA that are transmissible through the germline. Mutations in Nmnat1 were identified, and multiple outcrosses with wild-type mice served to eliminate most ENU-induced mutations at other chromosomal locations.31Wansleeben C. van Gurp L. Feitsma H. Kroon C. Rieter E. Verberne M. Guryev V. Cuppen E. Meijlink F. An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions.PLoS One. 2011; 6: e19357Crossref PubMed Scopus (26) Google Scholar One line harbors the p.V9M mutation, which was observed to cause LCA in patients from unrelated families and has been investigated in two separate studies.8Falk M.J. Zhang Q. Nakamaru-Ogiso E. Kannabiran C. Fonseca-Kelly Z. Chakarova C. Audo I. Mackay D.S. Zeitz C. Borman A.D. Staniszewska M. Shukla R. Palavalli L. Mohand-Said S. Waseem N.H. Jalali S. Perin J.C. Place E. Ostrovsky J. Xiao R. Bhattacharya S.S. Consugar M. Webster A.R. Sahel J.A. Moore A.T. Berson E.L. Liu Q. Gai X. Pierce E.A. NMNAT1 mutations cause Leber congenital amaurosis.Nat Genet. 2012; 44: 1040-1045Crossref PubMed Scopus (141) Google Scholar, 27Sasaki Y. Margolin Z. Borgo B. Havranek J.J. Milbrandt J. Characterization of Leber's congenital amaurosis-associated NMNAT1 mutants.J Biol Chem. 2015; 290: 17228-17238Crossref PubMed Scopus (29) Google Scholar The other line harbors a novel mutation, p.D243G, which has yet to be reported in the human population (ExAC Browser, http://exac.broadinstitute.org, accessed March 7, 2016). The retinal degenerative phenotype observed in both lines recapitulates key aspects of NMNAT1-LCA. These mouse models show promise for elucidating pathologic mechanisms associated with NMNAT1-LCA, developing therapies for patients with this disease, and understanding the roles of NMNAT1 in neuroprotection and NAD+ metabolism. Mice expressing the p.V9M variant of NMNAT1 were derived from a gene-driven screen of the Harwell ENU DNA archive to identify Nmnat1 missense alleles. The archive consists of pooled DNA samples from >10,000 ENU-mutagenized G1 male mice, which is paralleled by frozen sperm samples.29Coghill E.L. Hugill A. Parkinson N. Davison C. Glenister P. Clements S. Hunter J. Cox R.D. Brown S.D. A gene-driven approach to the identification of ENU mutants in the mouse.Nat Genet. 2002; 30: 255-256Crossref PubMed Scopus (155) Google Scholar, 32Quwailid M.M. Hugill A. Dear N. Vizor L. Wells S. Horner E. Fuller S. Weedon J. McMath H. Woodman P. Edwards D. Campbell D. Rodger S. Carey J. Roberts A. Glenister P. Lalanne Z. Parkinson N. Coghill E.L. McKeone R. Cox S. Willan J. Greenfield A. Keays D. Brady S. Spurr N. Gray I. Hunter J. Brown S.D. Cox R.D. A gene-driven ENU-based approach to generating an allelic series in any gene.Mamm Genome. 2004; 15: 585-591Crossref PubMed Scopus (138) Google Scholar Oligonucleotide primers (Table 1) were designed to allow amplification of all protein-coding sequences within Nmnat1 and used for PCR amplification of the pooled DNA samples. Amplified products were assessed by high-resolution melting curve analysis with the use of the LightScanner platform (Idaho Technology, Salt Lake City, UT). Because of the reduced melting temperature of mismatched nucleotides, ENU-induced mutations are evident as left-shifted melt curves, which are then verified by Sanger sequencing. This approach identified the Nmnat1 c.25G>A allele (Nmnat1imh, hereafter referred to as Nmnat1V9M), which was subsequently re-derived by in vitro fertilization using cryopreserved sperm (mixed BALB/c and C3H/HeH)33Thornton C.E. Brown S.D. Glenister P.H. Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.Mamm Genome. 1999; 10: 987-992Crossref PubMed Scopus (101) Google Scholar and wild-type C57BL/6J (B6) oocytes. The subsequent progeny were then bred into the wild-type B6 background. Before data collection, this mouse strain was outcrossed to B6 mice five times to eliminate approximately 97% of the ENU-induced mutations. On the basis of the mutation rate of approximately 0.67 mutations/Mbp for ENU mutagenesis and the size of the mouse genome (approximately 2.5 × 103 Mbp),31Wansleeben C. van Gurp L. Feitsma H. Kroon C. Rieter E. Verberne M. Guryev V. Cuppen E. Meijlink F. An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions.PLoS One. 2011; 6: e19357Crossref PubMed Scopus (26) Google Scholar it would be anticipated that in a mouse where no mutation is being selected for during outcrossing, only approximately 50 induced mutations would remain across the entire genome, of which just one would be expected to be in a coding region.34Keays D.A. Clark T.G. Flint J. Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens.Mamm Genome. 2006; 17: 230-238Crossref PubMed Scopus (53) Google Scholar The chance of one additional coding mutation that is physically linked to the known mutation on mouse chromosome 4 persisting after five outcrosses is 22%.34Keays D.A. Clark T.G. Flint J. Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens.Mamm Genome. 2006; 17: 230-238Crossref PubMed Scopus (53) Google Scholar To further mitigate the possibility of influence from the presence of an unidentified mutation, all experiments were performed on age-matched homozygous mutant Nmnat1V9M, heterozygous Nmnat1V9M, and wild-type mice generated by intercrossing heterozygous Nmnat1V9M mice.Table 1Set of Oligonucleotide Primer Pairs that Provide Complete Coverage of the Nmnat1 Protein-Coding Sequences for PCR Amplification and SequencingExonForwardReverse25′-GGTTGCATGTAGGTCAACAC-3′5′-GTCTTTAATTAGCTGGGTCGC-3′35′-TAAAGTCTGATTTGTTATGCCTAATATCG-3′5′-ACAAGAACATGTGGACTGC-3′45′-TCTGACATTAAGGAGTGTGCT-3′5′-TTTGGAGTCCTGGTAGACATC-3′55′-CCTGACCTTGTGCTTGATTC-3′5′-TGGTGGACGAGATGTCATT-3′55′-CAGAGCAACATCCACCT-3′5′-GAGTCTCCAGACGAGCC-3′These primers were used in the procedures that led to the identification of the p.V9M-Nmnat1 allele. Open table in a new tab These primers were used in the procedures that led to the identification of the p.V9M-Nmnat1 allele. The Nmnat1tvrm113 mutant, hereafter referred to as Nmnat1D243G, was identified in a B6 G3 ENU mutagenesis screen from the Translational Vision Research Models program30Won J. Shi L.Y. Hicks W. Wang J. Naggert J.K. Nishina P.M. Translational vision research models program.Adv Exp Med Biol. 2012; 723: 391-397Crossref PubMed Scopus (11) Google Scholar at The Jackson Laboratory (Bar Harbor, ME) by indirect ophthalmoscopy. The fundus examination revealed an abnormal retina with pigmentary changes and a grainy appearance with light spots, and this phenotype segregated in a monogenic, recessive manner. The genomic location of the mutation was determined by linkage analysis. Genomic DNA from progeny of an F2 intercross of the mutant strain with DBA/2J (DBA) was extracted as described.35Buffone G.J. Darlington G.J. Isolation of DNA from biological specimens without extraction with phenol.Clin Chem. 1985; 31: 164-165Crossref PubMed Scopus (173) Google Scholar DNA from 10 affected and 10 unaffected F2 offspring was pooled and assayed genome-wide with 48 simple sequence length polymorphic markers known to differ between B6 and DBA. Once a map position was identified on chromosome 4, it was refined in a high-resolution intercross that involved 765 F2 mice of the intercross described. To identify the causative mutation, a whole mouse exome library was constructed, using fragmented genomic DNA (1 μg) to a peak size of 300 bp by sonicating on low power for 30 seconds with power on, 30 seconds with power off for a total of 10 minutes using a Diagenode Bioruptor UCD-200TM-EX (Denville, NJ). The precapture paired end library was constructed with the TruSeq DNA Sample Preparation Kit (part number FC-121-100; Illumina, San Diego, CA) with no size selection step and 18 cycles of PCR. The precapture library was hybridized to the Roche NimbleGen Mouse Exome (Reference no. 9999042611) capture probe set (Roche NimbleGen, Madison, WI) according to the manufacturer's instructions. The sequencing library was subjected to real-time quantitative PCR, pooled with two similar libraries, and sequenced on a single lane of an Illumina HiSeq 2000 (Illumina) using a 2 × 100 bases (paired end) sequencing protocol. High-throughput sequence data were sorted with a local JAX Galaxy interface pipeline.36Blankenberg D. Von Kuster G. Coraor N. Ananda G. Lazarus R. Mangan M. Nekrutenko A. Taylor J. Galaxy: a web-based genome analysis tool for experimentalists.Curr Protoc Mol Biol. 2010; Chapter 19 (Unit 19.10.1–21)Crossref PubMed Scopus (1080) Google Scholar, 37Giardine B. Riemer C. Hardison R.C. Burhans R. Elnitski L. Shah P. Zhang Y. Blankenberg D. Albert I. Taylor J. Miller W. Kent W.J. Nekrutenko A. Galaxy: a platform for interactive large-scale genome analysis.Genome Res. 2005; 15: 1451-1455Crossref PubMed Scopus (1526) Google Scholar, 38Goecks J. Nekrutenko A. Taylor J. Galaxy TeamGalaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.Genome Biol. 2010; 11: R86Crossref PubMed Scopus (2670) Google Scholar Sequence reads were quality assessed using FastQC version 0.5 (Babraham Bioinformatics, Babraham, UK; http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and aligned to the mouse reference genome (mm10) from the University of California Santa Cruz, released December 2011, using BWA version 1.2.3.39Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26642) Google Scholar PCR duplicates were removed with SAMtools rmdup version 1.0.0.40Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31547) Google Scholar Single nucleotide polymorphisms and insertions/deletions were called with SAMtools mpileup version 1.0.0,40Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-
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