Obesity, non-insulin dependent diabetes mellitus (NIDDM) and heart disease are highly prevalent metabolic diseases that afflict a large proportion of the aging population in the United States. These diseases should be viewed as aspects of a metabolic syndrome that is produced by the interaction of many genes, rather than as separate entities. To illustrate the complexity of the issue, there are approximately 500 to 1,000 genes in mice that may lead to obesity when mutated. Our program focuses on identification of new obesity and type 2 diabetes mutations and their genetic modifiers.
We are investigating aspects of sensory loss in addition to the biochemical obesity and type 2 diabetes pathways in Alström and similar genetic syndromes. Our laboratory identified a human gene, ALMS1, that is mutated in patients with Alström syndrome, a rare inherited condition characterized by multiple disorders, including childhood obesity, retinal and cochlear (inner ear) degeneration, type 2 diabetes and hyperlipidemia (elevation of fats in the bloodstream, including cholesterol and triglycerides).
Mouse models of vision research have been instrumental in identifying primary mutations within genes that lead to ocular disorders and in identifying pathways important in retinal function through modifier screens, protein, and expression profiling. Our program is dedicated to identifying genes which when mutated lead to ocular diseases, to identify mechanisms underlying the function of those genes as well as the resulting pathological changes. The models are derived both from spontaneous mutations as well as chemical mutagenesis screens. Our lab focuses primarily on genes affecting the neural retina and supporting structures such as the retinal pigmented epithelium, glial cells and vascular network.
Genetic Interactions in Mouse Models of Human Diseases
Central regulation of energy homeostasis
Obesity, non-insulin dependent diabetes mellitus (NIDDM) and heart disease are highly prevalent metabolic diseases that afflict a large proportion of the aging population in the United States. Nearly 30 percent of adults are overweight or obese, about 10 percent of individuals over 65 have NIDDM, and 50 percent of deaths are caused by coronary artery disease. These diseases should be viewed as aspects of a metabolic syndrome that is produced by the interaction of many genes, rather than as separate entities. For example, only ~7 percent of obese patients develop NIDDM, whereas ~90 percent of NIDDM patients are obese. This suggests that in NIDDM patients, obesity, genes interact with diabetes susceptibility genes to produce the obese/diabetic phenotype. Obesity genes alone would only lead to obesity and diabetes susceptibility genes alone may not cause an overt phenotype. Further, to complicate the issue, one can estimate from the number of transgenic and targeted mouse models that develop an obese phenotype that there are 500 to 1,000 genes that when mutated will lead to obesity. Therefore, we believe that a broader knowledge of obesity pathways is necessary to design interventions with minimal side effects. To obtain additional entry points into obesity pathways, our program focuses on identification of new obesity and type 2 diabetes mutations and their genetic modifiers, and on how these mutations influence energy homeostasis to lead to the obese-diabetic phenotype.
TALLYHO, a new model for type 2 diabetes
Because type 2 diabetes occurs in the context of obesity, and insulin resistance genes have to interact with pancreas insufficiency genes to create the hyperglycemic phenotype, few mouse models of type 2 diabetes exist. We have established a new inbred strain of diabetic mice, TALLYHO/Jng (TH), through a backcross/intercross strategy with selection for male hyperglycemia. This new model of type 2 diabetes is characterized by moderate obesity, hyperinsulinemia, glucose intolerance and enlargement of the pancreatic islets of Langerhans. Male TH mice become overtly diabetic by 8 weeks of age. Breeding experiments suggested that the hyperglycemic trait was caused by a newly acquired autosomal recessive, single gene mutation that occurred on a permissive genetic background. In mapping crosses, we localized Tanidd1 (TallyHo associated non-insulin dependent diabetes mellitus 1), the major diabetes locus in the cross, to Chromosome 19 and constructed congenic lines to aid in fine mapping and cloning of the gene. Additional diabetes and obesity susceptibility genes segregate in a (TH x C57BL/6J)F1 x TH backcross. Furthermore, the Tanidd1 mutation epistatically interacts with loci on Chromosomes 6, 8, and 18. Physiological experiments indicate that insulin secretion is normal in male TH mice, but that TH mice show decreased glucose uptake in adipose tissue and muscle. The decreased glucose uptake is at least in part due to an abnormal cellular distribution of the facilitative glucose transporter SLC2A4 (formerly GLUT4) in adipocytes, and a failure of insulin to recruit SLC2A4 to the cell surface. The abnormal intracellular localization of SLC2A4 is also observed histochemically and by subcellular fractionation in the TH strain as well as in the Chromosome 19 congenic line B6.TH-Tanidd1TALLYHO/Jng, but not in the reverse congenic TH.CAST-Tanidd1CAST/Ei carrying the castaneus-derived wildtype allele of Tanidd1. Further experiments also indicated impairments in the insulin signaling pathway such as reduced activity of insulin receptor substrate 1 and reduced amounts of PI3 kinase. This suggests that Tanidd1 is affecting the signaling pathway leading to insulin-stimulated recruitment of SLC2A4.
Obesity-sensory loss syndromes
Several syndromes exist in the human population that are characterized by obesity, diabetes and loss of vision and hearing. Alström syndrome is a rare, recessive human disease of childhood obesity, retinal and cochlear degeneration and diabetes and heart disease. Alström syndrome and the phenotypically similar but genetically distinct Bardet-Biedl syndrome (BBS) show a remarkable phenotypic similarity with the tubby mouse. Because of this similarity and co-localization of the gene products in specific tissues (e.g., TUB and ALMS1 co-localize in the pancreatic alpha cells), as well as evidence that the underlying genes may function in intracellular transport (abnormal accumulation of vesicles is found in the inner segments of photoreceptor cells in Tub, Tulp1 and Alms1 mutant mice), we hypothesize that ALMS1, TUB, and the BBS proteins act in the same biochemical pathway.
Functional genomics of the tubby gene family
Tubby is an autosomal recessive mutation leading to a tripartite phenotype of maturity onset obesity, blindness, and deafness in B6(Cg)- Tubtub (B6-tub/tub) mice. We have shown that the obesity in tubby mice is not associated with hyperphagia or hypercorticism but with progressive insulin resistance. The progressive retinal degeneration in tubby mice is characterized by abnormal electroretinograms detected as early as 3 weeks of age and is caused by apoptotic loss of photoreceptor cells. Hearing loss is also apparent by 3 weeks of age and is characterized histologically by accelerated loss of outer hair cells and by progressive loss of inner hair cells. The obesity coupled with the retinal degeneration and hearing loss make tubby mice a good model for rare human monogenic disorders as described above. The Tub gene encodes a novel protein present in the cytoplasm and nucleoli of neuronal cell nuclei in the retina and brain. Tub is a member of a small gene family. We have identified the genes for three tubby-like proteins (Tulps) from mouse and human, respectively. Tulp1, when mutated in humans, causes retinitis pigmentosa 14 and leads to retinal degeneration in mice. Mutations in Tulp3 in the mouse lead to embryonic lethality and neural tube defects. An underlying abnormality in the knockout mice appears to be the apoptotic loss of ßIII-tubulin positive cells in the ventral neuroepithelium of the hindbrain. To search for biochemical pathways in which TUB plays a role, we carried out genetic modifier screens. We identified moth1, the modifier of tubby hearing 1, as the microtubule-associated protein 1A (Mtap1a). Mutations in the C57BL/6J (B6) allele of this gene lead to the hearing loss observed in B6-tub/tub mice. We also showed that these mutations reduce the binding of MTAP1A to members of the post-synaptic density (PSD) family of proteins, specifically DLG4 (formerly PSD95). PSDs are synaptic scaffolding proteins that link signaling components and the neuronal transport machinery. Our finding establishes a role for TUB in synapse function and suggests an interaction with the intracellular transport machinery. Further strengthening this notion is the identification of other transport-associated proteins such as myosin Vb and tropomodulin 2, as TULP binding partners, by yeast-two-hybrid (Y2H) analysis. Previously, little was known about the physiology of tubby mice. During testing of B6-tub/tub mice using the Comprehensive Lab Animal Monitoring System (CLAMS), we found tubby mice to have a lower respiratory quotient (RQ) compared to B6 controls, before the onset of obesity in both the light and the dark period (p In concordance with this data, tubby mice show a higher excretion of ketone bodies (7 weeks age, p p Quantitation of mRNA levels using real-time PCR showed that tubby mice fail to induce glucose-6-phosphate dehydrogenase (H6PD) during the transition from the light to the dark period (p In addition, several citric acid cycle genes are misregulated in tubby mice. The lipolytic enzymes acetyl-coA synthetase and carnitine palmitoyl transferase are increased during the dark cycle (ppi>tub mutation, since TUB is not expressed in liver. Examination of hypothalamic gene expression showed high levels of prepro-orexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. Based on our study and published reports on orexin action, we hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the obesity observed in tubby mice.
Through ascertainment and recombinational analyses of families, we mapped the Alström syndrome gene (ALMS1) to human chromosome 2p12, narrowed the minimal interval for ALMS1 to ~1.2 megabases, and through candidate gene testing identified a novel gene in which multiple mutations occurred in affected patients but not in unaffected controls. Mutation screening to date has identified major mutations such as nonsense mutations and insertions and deletions causing a frame shift and premature translation termination, suggesting that missense mutations do not cause disease or lead to a milder phenotype that is not recognized as Alström syndrome. Although Alström syndrome is a rare disease and mutations in the same gene may not play a role in more common forms of obesity, because an obese phenotype is observed, the gene still must be part of a pathway leading to obesity and identification of its functional role will contribute to our overall goal of describing the network of obesity pathways. Analysis of Alms1 gene expression and protein abundance showed that ALMS1 is present in all tissues affected in the disease. However, there is specific expression in the hypothalamus, for example in the parvicellular neurons of the paraventricular nucleus or the alpha cell of the pancreatic islets. We have found that human Alström patients, whether diabetic or not, have higher serum glucagon levels than either normal or type 2 diabetic controls. This may indicate that the diabetes in Alström is driven by abnormalities in glucagon secretion. To address the question as to whether diabetes in Alström could be driven by glucagon (as it is in conditions of pseudo-glucagonoma), we are currently measuring the glucagon and insulin response to glucose in isolated islets from wildtype and mutant mice. We have recently obtained homozygous offspring derived from gene-trapped mice that recapitulate common ALMS1 mutations in humans. Gratifyingly, the mutant mice show all the phenotypes observed in human patients and will be an invaluable model system for elucidating the biochemical function of the ALMS1 protein and studying the pathological consequences of its loss of function.
Novel single gene mouse obesity mutations
For the past several years, we have collected phenodeviants with increased body weight from the production and research animal colonies at The Jackson Laboratory. We are also mapping and cloning selected mutants from the Neuroscience Mutagenesis Facility and the Mouse Heart, Lung, Blood and Sleep Disorders Center at The Jackson Laboratory. We are actively pursuing four mutants (see table below). Domob1 and Nmf15 are in the fine-mapping stage, and positional candidate gene testing has begun.
|Mutant||Inheritance||Current Genetic Background||Map position Chr.||Phenotype|
|Domob1||dominant||B6||10||Slowly progressive morbid obesity|
|Nmf15||dominant||B6||5||Slowly progressive morbid obesity|
|fuldib1||recessive||Mixed*||4||Moderate obesity, rapid severe type 2 diabetes remutation of db, renamed Leprdb-od|
|hlb124||recessive||B6||Un||Increased body fat, near normal weight|
Our goal is to identify new entry points into obesity pathways through positional cloning of new obesity mutations, as we have done in the past with fat, tub, and Alms1, making the new models available to the research community. For our own continued investigation of these genes, we will focus on those that act in the CNS. We will begin to elucidate gene function through biochemical and physiological analyses, identifying additional key molecules in disease pathways through genetic modifier screens.
Molecular genetics of retinal development and maintenance
Approximately 50 million people worldwide are blind and ~150 million are significantly vision-impaired. Within the United States, ~3.8 million individuals over the age of 40 are blind or severely vision-impaired. Except for trauma and infections, the majority of human eye diseases are genetic in nature. A query of the Online Mendelian Inheritance in Man (OMIM) database using "retinal disease" as an identifier yields 353 entries. It is further estimated that the total number of retinal disease genes is 2-3 times the present figure of known or mapped genes (Wright & van Heyningen, 2001). Mouse models have been instrumental in the study of ocular disease that occurs as a result of heritable mutations in the human population. Initially, the goal of our research program was to use mouse models as an entry point to identify these molecules that were essential for normal retinal development and function through positional cloning efforts. With the maturation of our program, we have begun to focus on using these models to study gene function and mechanisms underlying disease pathology. The evolution of our program has come through the appreciation that genes do not act on their own but do so in the context of other genes. This means that a clinical outcome is not only dependent on a disease gene, but also on the genetic background in which it is found. Knowledge of modifiers and interaction partners is critically important in understanding the pathways that lead from a primary genetic defect to an observable phenotype. Further, genetic complexities are easier resolved in mice, and what we learn from mouse models is usually applicable to humans. The overriding theme of our program currently is the elucidation of interactions that occur among molecules to identify common functional pathways as well as pathways that lead to disease and are impacted by primary mutations. While the main approach of our program continues to be discovery research using genetic studies, we also have added a blend of marker analyses, functional studies, and generation of mouse resources that aim toward a greater understanding of the function and pathways in which the mutant retinal molecules we have identified act. During the past 12 years, we have discovered mutations in tubby (Tub), membrane frizzled-related protein (Mfrp), photoreceptor nuclear receptor (Nr2e3), destrin (Dstn), and Large (a putative glycosyltransferase) that all lead to ocular disease. We also identified two novel genes responsible for Alström (ALMS1) syndrome and retinitis pigmentosa in humans. Mutations in ALMS1 lead to cone dystrophy and early vision loss, hearing impairment, obesity and cardiomyopathy, while those in tubby like protein 1 (TULP1) lead to a rapid photoreceptor degeneration. In unpublished studies, we have identified the mutations in carbonic anhydrase, chloride channel 2, retinitis pigmentosa GTPase regulator interacting protein 1 (Rpgrip1), rhodopsin, phosphodiesterase 6A, prefoldin 5, c-mer proto-oncogene tyrosine kinase (Mertk), glutamate receptor, metabotropic 6 (Grm6), ubiqutin carboxyl-terminal esterase L3 (Uchl3), and nephrocystin-4 (Nphp4) that lead to either photoreceptor degeneration or CNS deficits. Finally, modifiers that suppress the hearing impairment in tubby mice were identified as mutations within Mtap1a, and those that enhance the retinal disease in Mfrprd6 mice were identified as a mutation in crumbs homolog1 (Crb1). As a result of these and previous efforts, and the use of database mining and transgenic approaches, we continue to work toward understanding the function of some of these proteins in the retina. We currently focus on molecules important in the development, function and maintenance of the 1) retinal pigmented epithelial cells; 2) external limiting membrane; 3) photoreceptor outer segments; and 4) Müller glial cells and astrocytes.
MFRP & C1QTNF5, dicistronic partners: Molecules important in establishing cellular adhesion & phagocytic function in the RPE?
The importance of the retinal pigment epithelium (RPE) to the health of the overlaying retina is well documented. The RPE serves a critical role in outer segment (OS) renewal. First, it serves to phagocytize and degrade proteins and lipids shed by photoreceptor OS discs, and second, it recycles chromophores necessary for the phototransduction cascade. The OS phagocytosis consists of a number of steps including recognition and binding, ingestion and formation of phagosomes, and degradation. These processes appear to be regulated by circadian rhythms and response to environmental light, such that the number of phagosomes in the RPE is highest shortly after the onset of light. One can imagine that loss of function of any of the gene products along this complex pathway could lead to severe consequences for the survival of the photoreceptor (PR) cells in the eye. The retinal degeneration 6 (rd6) mutant is an autosomal recessive mouse model of retinal degeneration that was first detected by indirect ophthalmoscopy with white spots that were observable as early as 8 weeks of age and exhibited a rapid photoreceptor degeneration. The disease-causing mutation, which mapped to Chromosome 9, was identified as a 4 bp deletion in a splice donor site in the Mfrp gene that leads to the skipping of exon 4. The deduced amino acid sequence of MFRP indicates a transmembrane domain, two cubilin domains, a low-density lipoprotein receptor a domain, and a cystine-rich domain with high homology to the frizzled family of proteins. Mfrp is proposed to express as a dicistronic transcript with C1q- and tumor necrosis factor-related protein 5 (C1qtnf5). C1QTNF5 is a small secreted protein containing a complement C1q domain. The Mfrp-C1qtnf5 dicistronic message is surprisingly expressed in the RPE cells and in the ciliary epithelium, as well as the lens, but not in photoreceptors. Based on preliminary studies, it appears that Mfrprd6 mice demonstrate abnormal RPE microvilli development, a delay in outer segment development, an impaired ability to phagocytize shed OSs, and potentially, an impairment of adhesion between RPE and outer segments. Using the Mfrprd6 model, and others with similar protein localization and phenotypes when mutated, we hope to gain a better understanding of how RPE cells affect photoreceptor development and the process of phagocytosis.
External limiting membrane (ELM) and its importance in retinal integrity
In the mammalian eye, apical-basal polarity is essential in PR cells for the transmission of light signals from the retina to the brain. One gene known to be important in maintaining apical-basal polarity and assisting in the formation of adherens junctions in the retina is Crumbs (Crb) , which encodes a transmembrane protein containing a long extracellular domain and a short C-terminal cytoplasmic tail. We have identified a mouse containing a single base pair deletion in the Crb1 gene. Clinically, Crb1rd8 develops retinal spotting in the inferior nasal quadrant by three weeks of age as demonstrated by indirect ophthalmoscopy. Histological evaluation reveals that these spots correspond with retinal dysplasia and pseudorosettes. In older mice, there is swelling and shortening of inner segments and a reduced number of outer segments in the areas affected by dysplasia. Immunohistochemical labeling for external limiting membrane (ELM) protein markers, including pan-cadherin, β-catenin, and PALS1 (MPP5), is fragmented or diffuse in these mutant mice, and electron microscopy demonstrates a reduced number of adherens junctions. Müller cell apical processes appear to be lost in regions lacking adherens junctions in these mice, as demonstrated by absent or reduced CD44 labeling and by electron microscopy. This observation suggests a developmental defect or the retraction of these processes. Interestingly, the phenotype seen in Crb1rd8 mutants varies when crossed onto different genetic backgrounds. In particular, when made congenic on the C57BL/6 background, retinal dysplasia is not observed. This phenotypic variation, which is similar to that seen in humans with CRB1 mutations, indicates that additional genetic factors are necessary for the dysplasia and subsequent focal PR degeneration to occur.
RPGRIP1 and interacting partners important in outer segment (OS) development
The photoreceptor cell of the retina is a neuronal cell comprised of an axon, the cell body containing the nucleus, the inner segment rich in mitochondria, and the outer segment, containing the phototransduction apparatus. Protein synthesis occurs in the inner segments and all proteins of the outer segments must be transported through the connecting cilium (CC) located between inner and outer segments. The outer segment is in fact considered to be a modified cilium and basal bodies are located at the base of the CC. Transport of proteins occurs via intraflagellar transport particles. Outer segment proteins, like opsin, are packaged in vesicles that bud off the endoplasmic reticulum and move toward the base of the CC where they fuse with the cell membrane and the protein cargo is tethered to the intraflagellar transport particle (IFT). The membrane bound cargo protein and the IFT are then moved by a kinesin motor along the microtubule axoneme of the CC to the OS. The IFT is returned to the inner segment via dynamin mediated transport. Mutations of any protein involved in these processes may impact OS development and maintenance. RPGRIP1 encodes the retinitis pigmentosa GTPase interacting protein 1 that contains a coiled-coil domain, a domain observed in proteins involved in vesicular trafficking, and a C-terminal RPGR interacting domain (RID). RPGRIP1 has been proposed by T. Li's group to be a structural component of the ciliary axoneme, acting as a scaffold for other proteins necessary for the transport of molecules down the coiled-coil. An insertional mutation into exon 14 of Rpgrip1 generated by Dr. Li's group initially developed a full complement of photoreceptors, but their outer segments were disorganized with grossly oversized outer segment discs that stack vertically, indicating the importance of RPGRIP1 in proper disc morphogenesis. We were somewhat surprised, therefore, when we identified a point mutation in the splice acceptor site of intron 6 within Rpgrip1 mutants that led to a deletion of exon 7, frame shift, and early termination, which had a more severe phenotype than the purported null mutant. Because the mutation was recessive in nature, it was unlikely to be a gain-of-function allele. Close examination of the original knockout construct/animal showed that it was an insertion of a neocassette into exon 14 that would likely lead to a frame shift and early termination. At the time the knockout was reported, it was suggested that no alternatively spliced variants existed for Rpgrip1 in mice. However, subsequently, it has been shown by Dr. Ferreira's group that a shorter form of Rpgrip1, an alternatively spliced variant does exist. The alternately spliced variant, Rpgrip1b, arises from the extension of exon 13 by three novel aa, and a premature truncation of RPGRIP1 that leads to the deletion of its C2 and RID domains. It is predominantly expressed in the retina. Therefore, it is likely that the original null is a hypomorph for the long form of Rpgrip1, whereas Rpgrip1nmf247 would be null or hypomorphic for both variants. We plan to examine the role of Rpgrip1b on OS initiation and morphogenesis. Nephrocystin-4 (NPHP4), a factor that directly interacts with RPGRIP1, belongs to a multifunctional complex that localizes to actin- and microtubule-based structures such as basal bodies and centrosomes of dividing cells. By in vivo and in vitro means, Dr. Roepman's group and others have shown that NPHP4 interacts with RPGRIP1 and nephrocystin, which in turn interacts with a host of other proteins, including but not limited to, filamin A-C, inversin, NPHP3, and tensin 1. Mutations within nephrocystin-4 are responsible for both nephronophthisis type 4 and Senior-Loken syndrome (SLNS4), a nephronophthisis associated with retinitis pigmentosa (RP). The age of onset and severity of RP is highly variable, ranging from congenital blindness, such as Leber's congenital amourosis (LCA), to late onset RP in these patients. It is suggested that the retinal disease in patients with SLNS4 is due to disruption of the interaction between RPGRIP1 and NPHP4. However, there is currently no genotype-phenotype relationship between mutations found in patients to the type of disease that manifests, indicating the potential for interaction with genetic background modifiers. We have recently identified a model carrying a mutation within the Nphp4 gene that leads to rapid photoreceptor degeneration as well as a host of other retinal defects. We will use the Rpgriplnmf247 and Nphp4 models to examine potential genetic interactions that occur in vivo.
Role of astrocytes and Müller glial cells in retinal vascular development
Considered as a group, developmental and acquired retinal vascular diseases are likely the leading cause of blindness and visual disability in the world. All ages are affected, from the newborn child to young adults to the elderly. The range of diseases associated with aberrant vascular changes include persistent fetal vasculature (PFV), retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), ischaemic and familial proliferative vitreoretinopathy (PVR), secondary epiretinal membranes, and the "wet" form of age-related macular degeneration. While much of the research focus has been placed on understanding the effects of angiogenic and anti-angiogenic molecules on endothelial cells, defects in other cell types may contribute to these vascular diseases. For example, astrocytes have an intimate association with developmental and acquired vascular growth. Astrocytes have also been shown to play a role in optic nerve development and maintenance, and in turn, normal retinal ganglion cell axonal projections are important for astrocyte migration, and therefore, can potentially affect retinal vascularization. It is likely that proteins involved in control of astrocyte differentiation, proliferation, migration, and maintenance are also involved in retinal neovascular processes as primary mechanisms or as susceptibility or resistance elements. Clearly, these are complex diseases that involve upstream and downstream agents that influence disease outcome. Defining molecular mechanisms for these diseases, which may be difficult in humans, is extremely important for development of appropriate and effective therapeutics. We have assembled a group of mutants--Nmf223, Lam1, Asvad1 and Coasvad1--that share similar clinical characteristics of occasional optic nerve colobomas, retinal vessel tortuosity, and vitreal vessels associated with fibroplasia. Common to all of these mutants is an abnormal retinal astrocytic network, suggesting underlying astrocytic defects. The availability of these mouse models that share similar phenotypic characteristics can be used to elucidate pathways important in disease development that go beyond the identification of a mutation in a single mutant. Identifying common pathways may make these disease phenotypes more accessible to drug/therapeutic agents.
Novel mutations and model development
We continue our quest to identify molecules important in retinal development and function by characterizing and identifying the underlying molecular bases for new spontaneous and chemically induced mutations. It should be noted that despite the availability of a fair number of retinal mutants, they represent a small fraction of the total retinal disease genes identified in humans. Also, in our mutagenesis facility, approximately 25 percent of the eye mutants evaluated represent novel loci. These models may allow us to flesh out the different pathways we are investigating above or provide additional entry points into pathways important in the maintenance of retinal function and integrity. We have screened ~7,500 G3 mice, identified 35 potential mutant lines that are undergoing heritability testing, and established heritability in 11 lines over the past year. Phenotypes range from ERG abnormalities to severe retinal degeneration. As we identify the underlying mutations in these mutants, they will be made available to the scientific community.
Common genetic suppressors of photoreceptor degeneration
Considering the large number of mutant genes that lead to retinal degeneration, therapeutics that affect pathways or mechanisms that are shared or "common" to all types of primary mutations may be the best targets. One pathway that appears to be associated with retinal and, potentially, macular degeneration is programmed cell death, or apoptosis. Previous studies have shown that mutations in genes such as Pde6b, Prph2, and Rho lead to apoptosis of photoreceptor cells. We have also shown that the degeneration in tub/tub and Tulp1-/- mutants is due to apoptosis. Little is known about what triggers programmed cell death in retinal degeneration, what the actual pathway is in the eye, or even if all mutations identified utilize the same apoptotic pathway. A current study underway in our lab is designed to address these issues. We have chosen three genetically determined mutants that exhibit different clinical phenotypes and/or rates of retinal degeneration. rd3 mutant mice develop pan-retinal degeneration by 8 weeks, tub (formerly rd5) homozygotes by 12 weeks, and rd7 homozygotes by 16 weeks of age. rd3 and tub mutants each exhibit a similar loss of pigment epithelium and patches of pigment deposits, whereas rd7 homozygotes present with small dots across the retina. Each of these mutations are being intercrossed with three genetically diverse strains: CAST/EiJ, a wild derived strain, and two standard inbred strains--AKR/J, a stock originating from Fürth, and NOD.NON-H2nb1, a congenic made from closely related Swiss-derived strains originating in Japan. Photoreceptor function in F2 progeny from each cross is being assessed by histological examination. Genome-wide scans with simple sequence length polymorphic markers are being carried out and quantitative trait loci (QTL) that delay or suppress retinal degeneration identified. Our results to date show that each strain is able to delay or suppress retinal degeneration of one or more of the mutants. We are identifying QTL traits that are overlapping between mutations within the same (e.g. , a modifier for both tub and rd7 exists on Chromosome 11 in the B6 x AKR intercross) or different crosses (a locus on Chromosome 9 prevents tub-induced photoreceptor loss in a B6 x CAST intercross, and rd7-induced photoreceptor loss in a B6/NOD.NON intercross). Multiple loci have been identified within each intercross, indicating the complex modification of the retinal degenerative phenotype. Results from the crosses described will allow us to dissect and separate the genetic factors that lead to phenotypic variability and may provide access into the pathways through which photoreceptor degeneration proceeds. In the future, we will complete the crosses and genome scans to identify the photoreceptor degeneration suppressors. Common loci will be isolated in the form of congenics and these will be used as reagents to identify the genes underlying the suppression. In the long term, coupling QTL analyses of these crosses with other molecular techniques-such as gene expression profiling to identify candidate genes-will be powerful complementary approaches. The QTL analyses will give us loci that are important in delaying or suppressing retinal degeneration and the gene expression profiling will provide potential candidate genes.
Research Scientist: Bo Chang, M.D.
Associate Research Scientists: Mark Krebs, Ph.D., Ramani Soundararajan, Ph.D., Lihong Zhao, Ph.D.
Research Assistants: Jeremy Charette, Ron Hurd, Wanda Jordan, Jieping Wang
Colony Coordinator: Lisa Stone
Laboratory Technician: Bernard FitzMaurice, Jr.
Research Administrative Assistant: Norma D. Buckley
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Edwards MM, Marin de Evsikova C, Collin GB, Gifford E, Wu J, Hicks WL, Whiting C, Varvel NH, Maphis N, Lamb BT, Naggert JK, Nishina PM, Peachey NS. 2010. Photoreceptor degeneration, azoospermia, leukoencephalopathy, and abnormal RPE cell function in mice expressing an early stop mutation in CLCN2. Invest Ophthalmol Vis Sci 51:3264-3272. PMCID: PMC2891478
Budzynski E, Gross AK, McAlear SD, Peachey NS, Shukla M, He F, Edwards M, Won J, Hicks WL, Wensel TG, Naggert JK, Nishina PM. 2010. Mutations of the opsin gene (Y102H and I307N) lead to light-induced degeneration of photoreceptors and constitutive activation of phototransduction in mice. J Biol Chem 285:14521-14533. PMCID: PMC2863193
Edwards MM, Mammadova-Bach E, Alpy F, Klein A, Hicks WL, Roux M, Simon-Assmann P, Smith RS, Orend G, Wu J, Peachey NS, Naggert JK, Lefebvre O, Nishina PM. 2010. Mutations in Lama1 disrupt retinal vascular development and inner limiting membrane formation. J Biol Chem 285:7697-7711. PMCID: PMC2844215
Won J, Gifford E, Smith RS, Yi H, Ferreira PA, Hicks WL, Li T, Naggert JK, Nishina PM. 2009. RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis. Hum Mol Genet 18:4329-4339. PMCID: PMC2766293
Matsumura H, Kano K, Marin de Evsikova CM, Young JA, Nishina PM, Naggert JK, Naito K. 2009. Transcriptome analysis reveals an unexpected role of a collagen tyrosine kinase receptor gene, Ddr2, as a regulator of ovarian function. Physiol Genomics 39:120-129. PMCID: PMC2765065
Wang Y, Nishina PM, Naggert J. 2009. Degradation of IRS1 leads to impaired glucose uptake in adipose tissue of the type 2 diabetes mouse model TALLYHO/Jng. J Endocrinol 203:65-74. PMCID: PMC2853731
Haider NB, Mollema N, Gaule M, Yuan Y, Sachs AJ, Nystuen AM, Naggert JK, Nishina PM. 2009. Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction. Exp Eye Res 89: 365-372. PMC2720439
Wang Y, Zheng Y, Nishina PM, Naggert JK. 2009. A new mouse model of metabolic syndrome and associated complications. J Endocrinol 202:17-28. PMCID: PMC2853727
Lee B, Kano K, Young J, John SW, Nishina PM, Naggert JK, Naito K. 2009. A novel ENU-induced mutation, peewee, causes dwarfism in the mouse. Mamm Genome 20:404-413.
Sakamoto K, McCluskey M, Wensel TG, Naggert JK, Nishina PM. 2009. New mouse models for recessive retinitis pigmentosa caused by mutations in the Pde6a gene. Hum Mol Genet 18: 178-192. PMCID: PMC2644649
Maddox DM, Vessey KA, Yarbrough GL, Invergo BM, Cantrell DR, Inayat S, Balannik V, Hicks WL, Hawes NL, Byers S, Smith RS, Hurd R, Howell D, Gregg RG, Chang B, Naggert JK, Troy JB, Pinto LH, Nishina PM, McCall MA. 2008. Allelic variance between GRM6 mutants, Grm6nob3 and Grm6nob4 results in differences in retinal ganglion cell visual responses. J Physiol 586: 4409-4424. PMCID: PMC2614010
Kano K, Marin de Evsikova CM, Young J, Wnek C, Maddatu TP, Nishina PM, Naggert JK. 2008. A novel dwarfism with gonadal dysfunction due to loss-of-function allele of the collagen receptor gene, Ddr2, in the mouse. Mol Endocrinol 22: 1866-1880. PMCID: PMC2505327
Won J, Smith RS, Peachey NS, Wu J, Hicks WL, Nishina PM. 2008. Membrance frizzled-related protein is necessary for the normal development and maintenance of photoreceptor outer segments. Vis Neurosci 25:563-574. PMCID: PMC2727655
Haider NB, Zhang W, Hurd R, Ikeda A, Nystuen AM, Naggert JK, Nishina PM. 2008. Mapping of genetic modifiers of Nr2e3 rd7/rd7 that suppress retinal degeneration and restore blue cone cells to normal quantity. Mamm Genome 19:145-154.
Malm E, Ponjavic V, Nishina PM, Naggert JK, Hinman EG, Andreasson S, Marshall JD, Moller C. 2008. Full-field electroretinography and marked variability in clinical phenotype of Alstrom syndrome. Arch Ophthalmol 126:51-57.