Olga Lozynska

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state “catch or present” model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS–Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

Background Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use. Methodology/Principal Findings We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells. Conclusions/Significance We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.

Translational regulation of utrophin by miRNAs

Background Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne Muscular Dystrophy (DMD) locus. Its regulation is of therapeutic interest as its overexpression can compensate for dystrophins absence in animal models of DMD. The tissue distribution and transcriptional regulation of utrophin have been characterized extensively, and more recently translational control mechanisms that may underlie its complex expression patterns have begun to be identified. Methodology/Principal Findings Using a variety of bioinformatic, molecular and cell biology techniques, we show that the muscle isoform utrophin-A is predominantly suppressed at the translational level in C2C12 myoblasts. The extent of translational inhibition is estimated to be ∼99% in C2C12 cells and is mediated by both the 5′- and 3′-UTRs of the utrophin-A mRNA. In this study we identify five miRNAs (let-7c, miR-150, miR-196b, miR-296-5p, miR-133b) that mediate the repression, and confirm repression by the previously identified miR-206. We demonstrate that this translational repression can be overcome by blocking the actions of miRNAs, resulting in an increased level of utrophin protein in C2C12 cells. Conclusions/Significance The present study has identified key inhibitory mechanisms featuring miRNAs that regulate utrophin expression, and demonstrated that these mechanisms can be targeted to increase endogenous utrophin expression in cultured muscle cells. We suggest that miRNA-mediated inhibitory mechanisms could be targeted by methods similar to those described here as a novel strategy to increase utrophin expression as a therapy for DMD.

Differential Expression of Utrophin-A and -B Promoters in the Central Nervous System (CNS) of Normal and Dystrophic mdx Mice

Utrophin (Utrn) is the autosomal homolog of dystrophin, the Duchene Muscular Dystrophy (DMD) locus product and of therapeutic interest, as its overexpression can compensate dystrophins absence. Utrn is transcribed by Utrn‐A and ‐B promoters with mRNAs differing at their 5′ ends. However, previous central nervous system (CNS) studies used C‐terminal antibodies recognizing both isoforms. As this distinction may impact upregulation strategies, we generated Utrn‐A and ‐B promoter‐specific antibodies, Taqman Polymerase chain reaction (PCR)‐based absolute copy number assays, and luciferase‐reporter constructs to study CNS of normal and dystrophic mdx mice. Differential expression of Utrn‐A and ‐B was noted in microdissected and capillary‐enriched fractions. At the protein level, Utrn‐B was predominantly expressed in vasculature and ependymal lining, whereas Utrn‐A was expressed in neurons, astrocytes, choroid plexus and pia mater. mRNA quantification demonstrated matching patterns of differential expression; however, transcription–translation mismatch was noted for Utrn‐B in caudal brain regions. Utrn‐A and Utrn‐B proteins were significantly upregulated in olfactory bulb and cerebellum of mdx brain. Differential promoter activity, mRNA and protein expressions were studied in cultured C2C12, bEnd3, neurons and astrocytes. Promoter activity ranking for Utrn‐A and ‐B was neurons > astrocytes >  C2C12 > bEnd3 and bEnd3 > astrocytes >  neurons > C2C12, respectively. Our results identify promoter usage patterns for therapeutic targeting and define promoter‐specific differential distribution of Utrn isoforms in normal and dystrophic CNS.

C. elegans dysferlin homolog fer-1 is expressed in muscle, and fer-1 mutations initiate altered gene expression of muscle enriched genes

Mutations in the human dysferlin gene cause Limb Girdle Muscular Dystrophy 2B (LGMD2B). The Caenorhabditis elegans dysferlin homolog, fer-1, affects sperms development but is not known to be expressed in or have a functional roles outside of the male germline. Using several approaches, we show that fer-1 mRNA is present in C. elegans muscle cells but is absent from neurons. In mammals, loss of muscle-expressed dysferlin causes transcriptional deregulation of muscle expressed genes. To determine if similar alterations in gene expression are initiated in C. elegans due to loss of muscle-expressed fer-1, we performed whole genome Affymetrix microarray analysis of two loss-of-function fer-1 mutants. Both mutants gave rise to highly similar changes in gene expression and altered the expression of 337 genes. Using multiple analysis methods, we show that this gene set is enriched for genes known to regulate the structure and function of muscle. However, these transcriptional changes do not appear to be in response to gross sarcomeric damage, since genetically sensitized fer-1 mutants exhibit normal thin filament organization. Our data suggest that processes other than sarcomere stability may be affected by loss of fer-1 in C. elegans muscle. Therefore, C. elegans may be an attractive model system in which to explore new muscle-specific functions of the dysferlin protein and gain insights into the molecular pathogenesis of LGMD2B.

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchennes muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over‐expression can compensate dystrophins absence. Utrophin is enriched at neuromuscular junctions due to heregulin‐mediated utrophin‐A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK‐dependent manner. MSK1/2 inhibition suppressed heregulin‐mediated utrophin‐A activation. MSK1 over‐expression potentiated heregulin‐mediated utrophin‐A activation and chromatin remodeling at the utrophin‐A promoter. These results identify MSK1/2 as key effectors modulating utrophin‐A expression as well as identify novel targets for DMD therapy.

On variants and disease-causing mutations: Case studies of a SEMA4A variant identified in inherited blindness

ABSTRACT The p.R713Q variant of the semaphorin‐4a‐encoding gene, SEMA4a, has been reported to cause autosomal dominant retinitis pigmentosa. Here we show three families with retinal degeneration in which unaffected family members are either homozygous or heterozygous for the variant. The p.R713Q variant in SEMA4A is insufficient to cause either autosomal recessive or autosomal dominant retinitis pigmentosa and is unlikely to be pathogenic.

Identification of a novel pathogenic missense mutation in PRPF31 using whole exome sequencing: a case report

Background Variants in PRPF31, which encodes pre-mRNA processing factor 31 homolog, are known to cause autosomal-dominant retinitis pigmentosa (adRP) with incomplete penetrance. However, the majority of mutations cause null alleles, with only two proven pathogenic missense mutations. We identified a novel missense mutation in PRPF31 in a family with adRP. Methods We performed whole exome sequencing to identify possible pathogenic mutations in the proband of a family with adRP. Available affected family members had a full ophthalmological evaluation including kinetic and two-colour dark adapted static perimetry, electroretinography and multimodal imaging of the retina. Two patients had evaluations covering nearly 20 years. We carried out segregation analysis of the probable mutation, PRPF31 c.590T>C. We evaluated the cellular localisation of the PRPF31 variant (p.Leu197Pro) compared with the wildtype PRPF31 protein. Results PRPF31 c.590T>C segregated with the disease in this four-generation autosomal dominant pedigree. There was intrafamilial variability in disease severity. Nyctalopia and mid-peripheral scotomas presented from the second to the fourth decade of life. There was severe rod >cone dysfunction. Visual acuity (VA) was relatively intact and was maintained until later in life, although with marked interocular asymmetries. Laboratory studies showed that the mutant PRPF31 protein (p.Leu197Pro) does not localise to the nucleus, unlike the wildtype PRPF31 protein. Instead, mutant protein resulted in punctate localisation to the cytoplasm. Conclusions c.590T>C is a novel pathogenic variant in PRPF31 causing adRP with incomplete penetrance. Disease may be due to protein misfolding and associated abnormal protein trafficking to the nucleus.

Prescreening whole exome sequencing results from patients with retinal degeneration for variants in genes associated with retinal degeneration

Background Accurate clinical diagnosis and prognosis of retinal degeneration can be aided by the identification of the disease-causing genetic variant. It can confirm the clinical diagnosis as well as inform the clinician of the risk for potential involvement of other organs such as kidneys. It also aids in genetic counseling for affected individuals who want to have a child. Finally, knowledge of disease-causing variants informs laboratory investigators involved in translational research. With the advent of next-generation sequencing, identifying pathogenic mutations is becoming easier, especially the identification of novel pathogenic variants. Methods We used whole exome sequencing on a cohort of 69 patients with various forms of retinal degeneration and in whom screens for previously identified disease-causing variants had been inconclusive. All potential pathogenic variants were verified by Sanger sequencing and, when possible, segregation analysis of immediate relatives. Potential variants were identified by using a semi-masked approach in which rare variants in candidate genes were identified without knowledge of the clinical diagnosis (beyond “retinal degeneration”) or inheritance pattern. After the initial list of genes was prioritized, genetic diagnosis and inheritance pattern were taken into account. Results We identified the likely pathogenic variants in 64% of the subjects. Seven percent had a single heterozygous mutation identified that would cause recessive disease and 13% had no obviously pathogenic variants and no family members available to perform segregation analysis. Eleven subjects are good candidates for novel gene discovery. Two de novo mutations were identified that resulted in dominant retinal degeneration. Conclusion Whole exome sequencing allows for thorough genetic analysis of candidate genes as well as novel gene discovery. It allows for an unbiased analysis of genetic variants to reduce the chance that the pathogenic mutation will be missed due to incomplete or inaccurate family history or analysis at the early stage of a syndromic form of retinal degeneration.