Characterization of Courtesy Stigma Perceived by Parents of Overweight Children with Bardet-Biedl Syndrome

Background

A child’s obesity is generally perceived by the public to be under the control of the child’s parents. While the health consequences of childhood obesity are well understood, less is known about psychological and social effects of having an obese child on parents. We set out to characterize stigma and courtesy stigma experiences surrounding obesity among children with Bardet-Biedl syndrome (BBS), a multisystem genetic disorder, and their parents.

Methods

Twenty-eight parents of children with BBS participated in semi-structured interviews informed by social stigmatization theory, which describes courtesy stigma as parental perception of stigmatization by association with a stigmatized child. Parents were asked to describe such experiences.

Results

Parents of children with BBS reported the child’s obesity as the most frequent target of stigmatization. They perceived health care providers as the predominant source of courtesy stigma, describing interactions that resulted in feeling devalued and judged as incompetent parents.

Conclusions

Parents of children with BBS feel blamed by others for their child’s obesity and described experiences that suggest health care providers may contribute to courtesy stigma and thus impede effective communication about managing obesity. Health care providers may reinforce parental feelings of guilt and responsibility by repeating information parents may have previously heard and ignoring extremely challenging barriers to weight management, such as a genetic predisposition to obesity. Strategies to understand and incorporate parents’ perceptions and causal attributions of their children’s weight may improve communication about weight control.     

Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis

Background

SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. I _Na dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.

Methods and Results

We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated I _Na was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like I _Na including peak I _Na density, activation and inactivation parameters, and late I _Na amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of I _Na late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.

Conclusion

The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Na_v1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.     

Cloning and characterization of a splice variant of human Bardet-Biedl syndrome 4 gene (BBS4).

Bardet-Biedl syndrome (BBS) is a heterogeneous multisystemic disorder characterized primarily by five cardinal features of retinal degeneration, obesity, polydactyly, hypogenitalism and mental retardation. To date, six distinct BBS loci that have been identified on different chromosomes. BBS4 gene is mapped to 15q22.2-23, which when mutated can cause BBS4. Its protein shows strong homology to O-linked N-acetylglucosamine (O-GlcNAc) transferase. Here we report a splice variant of BBS4, which is 2556 bp in length and has an open reading frame coding a predicted 527 amino-acids protein. RT-PCR shows that the cDNA is widely expressed while it has higher expression levels in pancreas, liver and prostate.     

Proteomic Interactions in the Mouse Vitreous-Retina Complex

Purpose

Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina.

Methods

Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software.

Results

We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor.

Conclusions

Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.     

Cadherin Expression,Vectorial Active Transport,and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells

Background

Cultures of human proximal tubule cells have been widely utilized to study the role of EMT in renal disease. The goal of this study was to define the role of growth media composition on classic EMT responses, define the expression of E- and N-cadherin, and define the functional epitope of MT-3 that mediates MET in HK-2 cells.

Methods

Immunohistochemistry, microdissection, real-time PCR, western blotting, and ELISA were used to define the expression of E- and N-cadherin mRNA and protein in HK-2 and HPT cell cultures. Site-directed mutagenesis, stable transfection, measurement of transepithelial resistance and dome formation were used to define the unique amino acid sequence of MT-3 associated with MET in HK-2 cells.

Results

It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. It was shown, based on the pattern of cadherin expression, connexin expression, vectorial active transport, and transepithelial resistance, that the HK-2 cell line has already undergone many of the early features associated with EMT. It was shown that the unique, six amino acid, C-terminal sequence of MT-3 is required for MT-3 to induce MET in HK-2 cells.

Conclusions

The results show that the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell. The HK-2 cell line, transfected with MT-3, may be an effective model to study the process of MET. The study implicates the unique C-terminal sequence of MT-3 in the conversion of HK-2 cells to display an enhanced epithelial phenotype.     

Hypothermia Protects and Prolongs the Tolerance Time of Retinal Ganglion Cells against Ischemia

Purpose

Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies.

Methods

Bovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed.

Results

Hypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia.

Conclusion

Hypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.     

Ectopic Expression of Human BBS4 Can Rescue Bardet-Biedl Syndrome Phenotypes in Bbs4 Null Mice

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogenitalism and renal defects. Recent findings have associated the etiology of the disease with cilia, and BBS proteins have been implicated in trafficking various ciliary cargo proteins. To date, 17 different genes have been reported for BBS among which BBS1 is the most common cause of the disease followed by BBS10, and BBS4. A murine model of Bbs4 is known to phenocopy most of the human BBS phenotypes, and it is being used as a BBS disease model. To better understand the in vivo localization, cellular function, and interaction of BBS4 with other proteins, we generated a transgenic BBS4 mouse expressing the human BBS4 gene under control of the beta actin promoter. The transgene is expressed in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. These mice were further bred to express the transgene in Bbs4 null mice, and their phenotype was characterized. Here we report that despite tissue specific variable expression of the transgene, human BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. These results provide an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies.     

LRP5 Is Required for Vascular Development in Deeper Layers of the Retina

Background

The low-density lipoprotein receptor-related protein 5 (LRP5) plays an important role in the development of retinal vasculature. LRP5 loss-of-function mutations cause incomplete development of retinal vessel network in humans as well as in mice. To understand the underlying mechanism for how LRP5 mutations lead to retinal vascular abnormalities, we have determined the retinal cell types that express LRP5 and investigated specific molecular and cellular functions that may be regulated by LRP5 signaling in the retina.

Methods and Findings

We characterized the development of retinal vasculature in LRP5 mutant mice using specific retinal cell makers and a GFP transgene expressed in retinal endothelial cells. Our data revealed that retinal vascular endothelial cells predominantly formed cell clusters in the inner-plexiform layer of LRP5 mutant retina rather than sprouting out or migrating into deeper layers to form normal vascular network in the retina. The IRES-β-galactosidase (LacZ) report gene under the control of the endogenous LRP5 promoter was highly expressed in Müller cells and was also weakly detected in endothelial cells of the retinal surface vasculature. Moreover, the LRP5 mutant mice had a reduction of a Müller cell-specific glutamine transporter, Slc38a5, and showed a decrease in b-wave amplitude of electroretinogram.

Conclusions

LRP5 is not only essential for vascular endothelial cells to sprout, migrate and/or anastomose in the deeper plexus during retinal vasculature development but is also important for the functions of Müller cells and retinal interneurons. Müller cells may utilize LRP5-mediated signaling pathway to regulate vascular development in deeper layers and to maintain the function of retinal interneurons.     

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

Background

Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.

Principle Findings

Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.

Conclusion

Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.