A cellular and molecular spatial atlas of dystrophic muscle.

Asynchronous skeletal muscle degeneration/regeneration is a hallmark feature of Duchenne muscular dystrophy (DMD); however, traditional -omics technologies that lack spatial context make it difficult to study the biological mechanisms of how asynchronous regeneration contributes to disease progression. Here, using the severely dystrophic D2-mdx mouse model, we generated a high-resolution cellular and molecular spatial atlas of dystrophic muscle by integrating spatial transcriptomics and single-cell RNAseq datasets. Unbiased clustering revealed nonuniform distribution of unique cell populations throughout D2-mdx muscle that were associated with multiple regenerative timepoints, demonstrating that this model faithfully recapitulates the asynchronous regeneration observed in human DMD muscle. By probing spatiotemporal gene expression signatures, we found that propagation of inflammatory and fibrotic signals from locally damaged areas contributes to widespread pathology and that querying expression signatures within discrete microenvironments can identify targetable pathways for DMD therapy. Overall, this spatial atlas of dystrophic muscle provides a valuable resource for studying DMD disease biology and therapeutic target discovery.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

Complement expression in the retina is not influenced by short-term pressure elevation.

PURPOSE: To determine whether short-term pressure elevation affects complement gene expression in the retina in vitro and in vivo. METHODS: Muller cell (TR-MUL5) cultures and organotypic retinal cultures from adult mice and monkeys were subjected to either 24-h or 72-h of pressure at 0, 15, 30, and 45 mmHg above ambient. C57BL/6 mice were subjected to microbead-induced intraocular pressure (IOP) elevation for 7 days. RNA and protein were extracted and used for analysis of expression levels of complement component genes and complement component 1, q subcomponent (C1q) and complement factor H (CFH) immunoblotting. RESULTS: mRNA levels of complement genes and C1q protein levels in Muller cell cultures remained the same for all pressure levels after exposure for either 24 or 72 h. In primate and murine organotypic cultures, pressure elevation did not produce changes in complement gene expression or C1q and CFH protein levels at either the 24-h or 72-h time points. Pressure-related glial fibrillary acidic protein (GFAP) mRNA expression changes were detected in primate retinal organotypic cultures (analysis of variance [ANOVA]; p<0.05). mRNA expression of several other genes changed as a result of time in culture. Eyes subjected to microbead-induced IOP elevation had no differences in mRNA expression of complement genes and C1q protein levels (ANOVA; p>0.05 for both) with contralateral control and naïve control eyes. CONCLUSIONS: Short-term elevation of pressure in vitro as well as short-term (1 week) IOP elevation in vivo does not seem to dramatically alter complement system gene expression in the retina. Prolonged expression to elevated pressure may be necessary to affect the complement system expression.

Primary cilia dynamics instruct tissue patterning and repair of corneal endothelium.

Primary cilia are required for several signaling pathways, but their function in cellular morphogenesis is poorly understood. Here we show that emergence of an hexagonal cellular pattern during development of the corneal endothelium (CE), a monolayer of neural crest-derived cells that maintains corneal transparency, depends on a precise temporal control of assembly of primary cilia that subsequently disassemble in adult corneal endothelial cells (CECs). However, cilia reassembly occurs rapidly in response to an in vivo mechanical injury and precedes basal body polarization and cellular elongation in mature CECs neighboring the wound. In contrast, CE from hypomorphic IFT88 mutants (Tg737(orpk)) or following in vivo lentiviral-mediated IFT88 knockdown display dysfunctional cilia and show disorganized patterning, mislocalization of junctional markers, and accumulation of cytoplasmic acetylated tubulin. Our results indicate an active role of cilia in orchestrating coordinated morphogenesis of CECs during development and repair and define the murine CE as a powerful in vivo system to study ciliary-based cellular dynamics.

Marfan syndrome caused by a novel FBN1 mutation with associated pigmentary glaucoma.

Mutations in fibrillin-1 (FBN1) cause a wide spectrum of disorders, including Marfan syndrome, which have in common defects in fibrillin-1 microfibrils. Ectopia lentis and myopia are frequently observed ocular manifestations of Marfan syndrome. Glaucoma is also associated with Marfan syndrome, though the form of glaucoma has not been well-characterized. In this report, ocular examination of a patient diagnosed with Marfan syndrome based on family history and aortic dilatation was performed, including measurement of facility of aqueous humor outflow by tonography. The patient did not have ectopia lentis at the age of 42 years. Based on optic nerve appearance, reduced outflow facility, elevated IOP with open angles and clear signs of pigment dispersion, the patient was diagnosed with pigmentary glaucoma. The patient was heterozygous for a novel truncating mutation in FBN1, p.Leu72Ter. Histology of normal human eyes revealed abundant expression of elastic fibers and fibrillin-1 in aqueous humor outflow structures. This is the first report of a patient with Marfan syndrome that is caused by a confirmed FBN1 mutation with associated pigmentary glaucoma. In addition to identifying a novel mutation of FBN1 and broadening the spectrum of associated ocular phenotypes in Marfan syndrome, our findings suggest that pigmentary glaucoma may involve defects in fibrillin-1 microfibrils.

Multiancestry exome sequencing reveals INHBE mutations associated with favorable fat distribution and protection from diabetes.

Body fat distribution is a major, heritable risk factor for cardiometabolic disease, independent of overall adiposity. Using exome-sequencing in 618,375 individuals (including 160,058 non-Europeans) from the UK, Sweden and Mexico, we identify 16 genes associated with fat distribution at exome-wide significance. We show 6-fold larger effect for fat-distribution associated rare coding variants compared with fine-mapped common alleles, enrichment for genes expressed in adipose tissue and causal genes for partial lipodystrophies, and evidence of sex-dimorphism. We describe an association with favorable fat distribution (p = 1.8 × 10-09), favorable metabolic profile and protection from type 2 diabetes (~28% lower odds; p = 0.004) for heterozygous protein-truncating mutations in INHBE, which encodes a circulating growth factor of the activin family, highly and specifically expressed in hepatocytes. Our results suggest that inhibin ßE is a liver-expressed negative regulator of adipose storage whose blockade may be beneficial in fat distribution-associated metabolic disease.

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.

Gene expression changes in steroid-induced IOP elevation in bovine trabecular meshwork.

PURPOSE: To determine whether gene expression changes occur in the trabecular meshwork (TM) of cow eyes with steroid-induced intraocular pressure (IOP) elevation. METHODS: Adult female Braford cows (n = 4) were subjected to uniocular prednisolone acetate treatment for 6 weeks. IOP was monitored with an applanation tonometer. At the conclusion of the experiment, animals were euthanized, eyes were enucleated, and the TM was dissected and stored in an aqueous nontoxic tissue storage reagent. RNA was extracted and subjected to microarray analysis using commercial oligonucleotide bovine arrays. Some of the genes differentially expressed between control and experimental eyes were confirmed by quantitative RT-PCR and some of the respective proteins were studied by immunoblotting. RESULTS: IOP began to increase after 3 weeks of treatment, reaching a peak 2 weeks later. IOP differences between corticosteroid-treated and fellow control eyes were 6 ± 1 mm Hg (mean ± SD) at the conclusion of the study. Microarray analysis revealed that expression of 258 genes was upregulated, whereas expression of 187 genes was downregulated in the TM of eyes with steroid-induced IOP elevation. Genes identified to be differentially expressed include genes coding for cytoskeletal proteins, enzymes, growth and transcription factors, as well as extracellular matrix proteins and immune response proteins. A number of relevant gene networks were detected by bioinformatic analysis. CONCLUSIONS: Steroid-induced IOP elevation alters gene expression in the bovine TM. Identification of genes with changing expression in this model of open-angle glaucoma may help elucidate the primary changes occurring at the molecular level in this condition.

Intraoperative floppy iris syndrome: report of a case and histopathologic analysis.

OBJECTIVE: To understand the role of the α(1A)-adrenoreceptors (ARs) in the pathophysiologic mechanism of intraoperative floppy iris syndrome (IFIS). METHODS: Iris specimens from a patient with tamsulosin hydrochloride-induced IFIS were obtained during trabeculectomy. Specimens underwent histological analysis and immunohistochemical analysis with antibodies specific for actin, myoglobin, α(1A)-ARs, and myosin. Iris specimens from a patient without IFIS were used for comparison. Samples were processed for transmission electron microscopy. RESULTS: Histological examination showed normal dilator muscle, arterioles, stroma, and pigment epithelium. Actin, myosin, and myoglobin distribution and intensities were similar between IFIS and non-IFIS tissue. The staining pattern and colocalization with myosin suggested that α(1A)-ARs are present in iris arteriolar muscularis in addition to the dilator muscle in both IFIS and control irides. Significantly less staining of IFIS tissue was found compared with the non-IFIS iris. Ultrastructures of melanocytes and stroma appeared to be normal. Iris arterioles possessed thick endothelial basement membranes, semilongitudinally oriented muscularis, and abundant perivascular collagen coats. CONCLUSIONS: We confirm the presence of α(1A)-ARs in human iris by results of immunohistochemical analysis. The α(1A)-ARs localize to iris arteriolar muscularis in addition to the iris dilator muscle. This localization suggests that IFIS may develop because of iris vascular dysfunction and that iris vasculature may have structural in addition to nutritive functions.

Gene expression changes in areas of focal loss of retinal ganglion cells in the retina of DBA/2J mice.

Purpose. To determine whether differences in gene expression occur between areas of focal retinal ganglion cell (RGC) loss and of relative RGC preservation in the DBA/2 mouse retina and whether they can provide insight into the pathophysiology of glaucoma. Methods. Areas of focal RGC loss (judged by lack of Fluorogold labeling; Fluorochrome, Denver, CO), adjacent areas with relative RGC preservation in DBA/2 retina, and Fluorogold-labeled retina from DBA/2(-pe) (pearl) mice were dissected and used for microarray analysis. RT-PCR and immunoblot analysis were used to confirm differential gene expression. Bioinformatic analysis was used to identify gene networks affected in the glaucomatous retina. Results. Microarray analysis identified 372 and 115 gene chip IDs as up- and downregulated, respectively, by 0.5-fold in areas of RGC loss. Differentially expressed genes included those coding for cytoskeletal proteins, enzymes, transport proteins, extracellular matrix (ECM) proteins, and immune response proteins. Several genes were confirmed by RT-PCR. For at least two genes, differential protein expression was verified. Bioinformatics analysis identified multiple affected functional gene networks. Pearl mice appeared to have significantly different gene expression, even when compared with relatively preserved areas of the DBA/2 retina. Conclusions. Regional gene expression changes occur in areas of focal RGC loss in the DBA/2 retina. The genes involved code for proteins with diverse cellular functions. Further investigation is needed to determine the cellular localization of the expression of these genes during the development of spontaneous glaucoma in the DBA/2 mouse and to determine whether some of these gene expression changes are causative or protective of RGC loss.