Microarray analysis of iris gene expression in mice with mutations influencing pigmentation.

PURPOSE: Several ocular diseases involve the iris, notably including oculocutaneous albinism, pigment dispersion syndrome, and exfoliation syndrome. To screen for candidate genes that may contribute to the pathogenesis of these diseases, genome-wide iris gene expression patterns were comparatively analyzed from mouse models of these conditions. METHODS: Iris samples from albino mice with a Tyr mutation, pigment dispersion-prone mice with Tyrp1 and Gpnmb mutations, and mice resembling exfoliation syndrome with a Lyst mutation were compared with samples from wild-type mice. All mice were strain (C57BL/6J), age (60 days old), and sex (female) matched. Microarrays were used to compare transcriptional profiles, and differentially expressed transcripts were described by functional annotation clustering using DAVID Bioinformatics Resources. Quantitative real-time PCR was performed to validate a subset of identified changes. RESULTS: Compared with wild-type C57BL/6J mice, each disease context exhibited a large number of statistically significant changes in gene expression, including 685 transcripts differentially expressed in albino irides, 403 in pigment dispersion-prone irides, and 460 in exfoliative-like irides. CONCLUSIONS: Functional annotation clusterings were particularly striking among the overrepresented genes, with albino and pigment dispersion-prone irides both exhibiting overall evidence of crystallin-mediated stress responses. Exfoliative-like irides from mice with a Lyst mutation showed overall evidence of involvement of genes that influence immune system processes, lytic vacuoles, and lysosomes. These findings have several biologically relevant implications, particularly with respect to secondary forms of glaucoma, and represent a useful resource as a hypothesis-generating dataset.

Spectral analysis by XANES reveals that GPNMB influences the chemical composition of intact melanosomes.

GPNMB is a unique melanosomal protein. Unlike many melanosomal proteins, GPNMB has not been associated with any forms of albinism, and it is unclear whether GPNMB has any direct influence on melanosomes. Here, melanosomes from congenic strains of C57BL/6J mice mutant for Gpnmb are compared to strain-matched controls using standard transmission electron microscopy and synchrotron-based X-ray absorption near-edge structure analysis (XANES). Whereas electron microscopy did not detect any ultrastructural changes in melanosomes lacking functional GPNMB, XANES uncovered multiple spectral phenotypes. These results directly demonstrate that GPNMB influences the chemical composition of melanosomes and more broadly illustrate the potential for using genetic approaches in combination with nano-imaging technologies to study organelle biology.

Elevated oxidative membrane damage associated with genetic modifiers of Lyst-mutant phenotypes.

LYST is a large cytosolic protein that influences the biogenesis of lysosome-related organelles, and mutation of the encoding gene, LYST, can cause Chediak-Higashi syndrome. Recently, Lyst-mutant mice were recognized to also exhibit an iris disease resembling exfoliation syndrome, a common cause of glaucoma in humans. Here, Lyst-mutant iris phenotypes were used in a search for genes that influence Lyst pathways. In a candidate gene-driven approach, albino Lyst-mutant mice homozygous for a mutation in Tyr, whose product is key to melanin synthesis within melanosomes, exhibited complete rescue of Lyst-mutant iris phenotypes. In a genetic background-driven approach using a DBA/2J strain of congenic mice, an interval containing Tyrp1 enhanced Lyst-dependent iris phenotypes. Thus, both experimental approaches implicated the melanosome, an organelle that is a potential source of oxidative stress, as contributing to the disease phenotype. Confirming an association with oxidative damage, Lyst mutation resulted in genetic context-sensitive changes in iris lipid hydroperoxide levels, being lowest in albino and highest in DBA/2J mice. Surprisingly, the DBA/2J genetic background also exposed a late-onset neurodegenerative phenotype involving cerebellar Purkinje-cell degeneration. These results identify an association between oxidative damage to lipid membranes and the severity of Lyst-mutant phenotypes, revealing a new mechanism that contributes to pathophysiology involving LYST.

Lyst mutation in mice recapitulates iris defects of human exfoliation syndrome.

PURPOSE: Human eyes with exfoliation syndrome (XFS) exhibit a distinctive pattern of iris transillumination defects that are recapitulated in Lyst mutant mice carrying the beige allele. The purpose of this study was to determine the anatomic basis for Lyst-mediated transillumination defects, test whether Lyst mutant mice develop other features of XFS, and describe the molecular basis of the beige mutation. METHODS: Lyst mutant mice and strain-matched controls were compared by clinical, histologic, immunohistochemical, and molecular genetic analyses. RESULTS: Slit-lamp examination showed that Lyst mutant mice uniformly exhibit XFS-like transillumination defects. Histologic analysis showed that these defects correlate with a sawtooth morphology of the iris pigment epithelium. Lyst mutant mice also produce an exfoliative-like material and exhibit pronounced pigment dispersion. Despite these insults, Lyst mutation does not cause increased intraocular pressure or optic nerve damage in the C57BL/6J genetic background. Sequence analysis identified that the beige mutation is predicted to delete a single isoleucine from the WD40 domain of the LYST protein, suggesting that this mutation is likely to disrupt a protein-protein interaction. CONCLUSIONS: Lyst mutant eyes exhibit multiple features of XFS. Recent human genetic association studies have identified changes occurring in the LOXL1 gene as an important risk factor for XFS but also indicated that other factors contributing to risk likely exist. These results demonstrated that mutation of the Lyst gene can produce ocular features of human XFS and suggested that LYST or LYST-interacting genes may contribute to XFS.

Iris phenotypes and pigment dispersion caused by genes influencing pigmentation.

Spontaneous mutations altering mouse coat colors have been a classic resource for discovery of numerous molecular pathways. Although often overlooked, the mouse iris is also densely pigmented and easily observed, thus representing a similarly powerful opportunity for studying pigment cell biology. Here, we present an analysis of iris phenotypes among 16 mouse strains with mutations influencing melanosomes. Many of these strains exhibit biologically and medically relevant phenotypes, including pigment dispersion, a common feature of several human ocular diseases. Pigment dispersion was identified in several strains with mutant alleles known to influence melanosomes, including beige, light, and vitiligo. Pigment dispersion was also detected in the recently arising spontaneous coat color variant, nm2798. We have identified the nm2798 mutation as a missense mutation in the Dct gene, an identical re-occurrence of the slaty light mutation. These results suggest that dysregulated events of melanosomes can be potent contributors to the pigment dispersion phenotype. Combined, these findings illustrate the utility of studying iris phenotypes as a means of discovering new pathways, and re-linking old ones, to processes of pigmented cells in health and disease.

GpnmbR150X allele must be present in bone marrow derived cells to mediate DBA/2J glaucoma.

BACKGROUND: The Gpnmb gene encodes a transmembrane protein whose function(s) remain largely unknown. Here, we assess if a mutant allele of Gpnmb confers susceptibility to glaucoma by altering immune functions. DBA/2J mice have a mutant Gpnmb gene and they develop a form of glaucoma preceded by a pigment dispersing iris disease and abnormalities of the immunosuppressive ocular microenvironment. RESULTS: We find that the Gpnmb genotype of bone-marrow derived cell lineages significantly influences the iris disease and the elevation of intraocular pressure. GPNMB localizes to multiple cell types, including pigment producing cells, bone marrow derived F4/80 positive antigen-presenting cells (APCs) of the iris and dendritic cells. We show that APCs of DBA/2J mice fail to induce antigen induced immune deviation (a form of tolerance) when treated with TGFbeta2. This demonstrates that some of the immune abnormalities previously identified in DBA/2J mice result from intrinsic defects in APCs. However, the tested APC defects are not dependent on a mutant Gpnmb gene. Finally, we show that the Gpnmb mediated iris disease does not require elevated IL18 or mature B or T lymphocytes. CONCLUSION: These results establish a role for Gpnmb in bone marrow derived lineages. They suggest that affects of Gpnmb on innate immunity influence susceptibility to glaucoma in DBA/2J mice.

Motility of a biflagellate sperm: waveform analysis and cyclic nucleotide activation.

The sperm of the freshwater clam Corbicula fluminea are unusual in that they have two flagella, both of which are capable of beating. When Corbicula sperm are removed from the gonad and placed into freshwater, most remain immotile. Video microscopy was used to assess signaling molecules capable of activating Corbicula sperm motility. Experiments using the cAMP analogs dbcAMP or 8-Br-cAMP show that elevating cAMP activates flagellar motility. Treatments with 8-Br-cGMP activated motility in similar numbers of sperm. Treatments with the selective cAMP-dependent protein kinase (PKA) inhibitor H-89 block activation by 8-Br-cAMP but not by 8-Br-cGMP. Similar treatments with the cGMP-dependent protein kinase (PKG) inhibitor Rp-8-pCPT-cGMPS block activation by 8-Br-cGMP but not by 8-Br-cAMP. These results suggest that cAMP and cGMP each work through their specific kinase to activate flagellar motility. Analysis of spontaneously activated freely swimming sperm shows that the two flagella beat with different parameters. The A flagellum beats with a shorter wavelength and a higher frequency than the B flagellum. The observed differences in flagellar waveform indicate that the flagella are differentially controlled.