Investigates the genetics of metabolic syndrome and ciliopathies.
Our research seeks to identify new metabolic syndrome (obesity and type 2 diabetes) mutations and their genetic modifiers and to determine how the underlying mutations cause the disease phenotype. We also investigate ciliopathies, diseases caused by impaired ciliary function that combine aspects of metabolic syndrome with sensory loss.
Because type 2 diabetes occurs in the context of obesity, and insulin resistance genes have to interact with pancreatic insufficiency genes to create the hyperglycemic phenotype, few mouse models of type 2 diabetes exist. We have established an 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. Further experiments also indicated impairments in the insulin-signaling pathway such as reduced levels of insulin receptor substrate 1 and reduced activations of PI3 kinase. This suggests that Tanidd1 is affecting the signaling pathway leading to insulin-stimulated recruitment of SLC2A4.
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 are ciliary proteins and 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.
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. 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. It has since been shown by others that the TULPs are ciliary proteins that are involved in transport events associated with G-coupled receptor signaling. In particular TULP3 was shown to act as an inhibitor of sonic hedgehog signaling by recruiting GPR161 to primary cilia.
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 compared to B6 controls, before the onset of obesity in both the light and the dark period. In concordance with this data, tubby mice show a higher excretion of ketone bodies. Quantitation of mRNA levels and enzymatic assays demonstrated that tubby mice do not use glycolysis for energy production during the dark cycle but instead rely on fatty acids as energy source. That indicates that pathways that are protective against obesity are intact in tubby mice. 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 establishes an elevated sympathetic tone, which in turn leads to changes in liver carbohydrate metabolism and may contribute to the late onset and mild obesity observed in tubby mice.
Alström syndrome (AS, OMIM #203800) a rare and severe recessive disorder, is characterized by chronic, debilitating pathological conditions that are also frequently observed in the general population. Most of the major organ systems are affected, and there is currently no treatment available. Phenotypes include progressive neurosensory retinal and aural degeneration, and obesity and Type II diabetes with their associated co-morbidities. We have identified the molecular basis of AS as mutations in a single gene whose protein product, ALMS1, localizes to centrosomes and ciliary basal bodies, implicating AS as one of a large number of ciliary diseases. While we have access to autopsy tissue from affected individuals that has provided significant insight to end-stage disease pathology, the entire spectrum of phenotypes and pathological progression of AS is poorly understood. Our laboratory has contributed significantly to the recruitment of patients, to collection of data and blood and tissue samples. We have generated Alms1 animal models, and we are a major source for reagents for the Alström research community. The availability of mouse models that recapitulate many of the AS phenotypes will contribute to the identification of the disease etiology in different organs, and may lead to the identification of possible points of therapeutic intervention.
AS is a member of a growing class of mostly severe, syndromic, single-gene diseases, the ciliopathies (Waters and Beales, 2011). Currently more than 50 diseases are known in which mutations in ciliary proteins cause a wide range of disease phenotypes (Davis and Katsanis, 2012). The common cause is the malfunction of the primary cilium, the signaling center of differentiated cells. Critical for elaborating the primary cilium are the basal bodies, microtubule-based structures that also serve as the microtubule organizing center of the cell and coordinate the intracellular trafficking events to and from the primary cilium. In dividing cells the basal bodies serve as centrosomes to assemble the spindle apparatus and initiate cytokinesis. Frequent in the ciliopathies is an involvement of the retina, to a large extent because the photoreceptor outer segment is a modified primary cilium, and a functioning transport through the connecting cilium is critical for photoreceptor health.
Mutation screening in AS 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 AS. Analysis of Alms1 gene expression and protein abundance showed that ALMS1 is present in all tissues affected in the disease. The Alms1 gene produces several alternately spliced transcripts, and we hypothesize that the proteins produced from these transcripts carry independent functions in the cell. ALMS1 proteins carrying an N-terminal epitope have been shown to localize to ciliary basal bodies where they bind to CNAP1. AMLS1 proteins containing a C-terminal epitope co-localize with endosomal structures, and yeast two hybrid analysis demonstrated interaction with other proteins that have been previously associated with the endosomal recycling pathway.
Ongoing studies are aimed at further defining the functions of ALMS1 in cells and at elucidating the disease pathways that are activated by Alms1 mutations. For example, because ALMS1 mutations reduce endosome recycling, TGFß signaling is increased in affected tissues and drives the severe fibrosis that develops in many tissues in AS patients.
A particular interest of our lab is the function of genetic modifier loci, and we have identified Alms1 modifiers that increase the severity of the retinal degeneration as well as a modifier that ameliorates the liver fibrosis in Alms1-deficient mice. We are currently testing hypotheses about the mechanisms through which these modifiers affect the disease process. We also carry out ENU mutagenesis screens to identify additional modifier genes.