Bruno 1/CELF regulates splicing and cytoskeleton dynamics to ensure correct sarcomere assembly in Drosophila flight muscles.

Muscles undergo developmental transitions in gene expression and alternative splicing that are necessary to refine sarcomere structure and contractility. CUG-BP and ETR-3-like (CELF) family RNA-binding proteins are important regulators of RNA processing during myogenesis that are misregulated in diseases such as Myotonic Dystrophy Type I (DM1). Here, we report a conserved function for Bruno 1 (Bru1, Arrest), a CELF1/2 family homolog in Drosophila, during early muscle myogenesis. Loss of Bru1 in flight muscles results in disorganization of the actin cytoskeleton leading to aberrant myofiber compaction and defects in pre-myofibril formation. Temporally restricted rescue and RNAi knockdown demonstrate that early cytoskeletal defects interfere with subsequent steps in sarcomere growth and maturation. Early defects are distinct from a later requirement for bru1 to regulate sarcomere assembly dynamics during myofiber maturation. We identify an imbalance in growth in sarcomere length and width during later stages of development as the mechanism driving abnormal radial growth, myofibril fusion, and the formation of hollow myofibrils in bru1 mutant muscle. Molecularly, we characterize a genome-wide transition from immature to mature sarcomere gene isoform expression in flight muscle development that is blocked in bru1 mutants. We further demonstrate that temporally restricted Bru1 rescue can partially alleviate hypercontraction in late pupal and adult stages, but it cannot restore myofiber function or correct structural deficits. Our results reveal the conserved nature of CELF function in regulating cytoskeletal dynamics in muscle development and demonstrate that defective RNA processing due to misexpression of CELF proteins causes wide-reaching structural defects and progressive malfunction of affected muscles that cannot be rescued by late-stage gene replacement.

Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration.

Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.

The PKG Inhibitor CN238 Affords Functional Protection of Photoreceptors and Ganglion Cells against Retinal Degeneration.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

Emotion regulation and social responsiveness in adults with autism spectrum disorder.

Objectives: This study aimed to find out the relationship between emotion regulation (ER) and its domains with social responsiveness (SR) to investigate ER and its domains as predictors of SR. Materials and Methods: A sample of 60 male and female adults diagnosed by a professional with autism spectrum disorder (ASD) was studied with ER and its domains of RI or Cognitive Reappraisal, SI or Expressive Suppression and SR as variables. Tools used were Social Responsiveness Scale-2 (Adult, Relative/Other online form) and Emotion Regulation Questionnaire (ERQ). Results: ERQ domain of Cognitive Reappraisal or RI was found to be negatively correlated with Social Responsiveness or SR but positively correlated with Expressive Suppression or SI domain with Pearson's r value of -0.662 for RI and of 0.275 for SI. Furthermore, RI and SI variables were found to be significantly negatively correlated with each other. Multiple regression analysis results showed R to be 0.666 and predictor variables explained 44.4% of the variance in the data since R square was found to be 0.444. The model was found to be a significant predictor of the variable SR, F (2, 57) = 22.76, P = 0.000. Conclusion: The present study found that ASD adults with high or good SR engage in less cognitive reappraisal (RI) ER strategy and more in expressive suppression (SI) strategy of ER. Multiple regression analysis results suggest a good and strong relationship suggesting our model is a relatively good predictor of the outcome.

Influence of Domestic Exposure to Polycyclic Aromatic Hydrocarbons on Women's Periconceptional Stage and Associated Risk of Oral Cleft in Offspring.

Background: Polycyclic aromatic hydrocarbons (PAHs) constitute a group of chemicals with an omnipresence in the environment and our surroundings. With their genotoxicity and carcinogenic nature, it has been proven to be monstrous in our daily life and, especially for pregnant women and their newborn. Aim: This questionnaire study was done to verify the influence of domestic exposure to polyaromatic hydrocarbons on women's periconceptional stage and risk of oral cleft in offspring in the suburban and the rural population of Mysore. Methodology: Two hundred pregnant women as patients from four different hospitals in Mysore were given a questionnaire to be filled with 24 parameters ranging from the knowledge to various means of exposure to the pregnant women with the PAH and the severity and the extent of the orofacial defect in the newborn. Results: It was determined that exposure of pregnant women to the smoke emanating from the method of cooking or heating to smoking (first or passive) and the direct inhalation of gas had the maximum effects on the association of cleft palate (60.7%) in unilateral followed by 90.9% in bilateral, 65.0% in soft tissue, and 76.2% in hard tissue cleft palate. Conclusion: The deleterious effects of the cooking and water heating measures practiced in the suburban and the rural population predisposed the pregnant women to significantly higher chances of offspring with the varied extent of the orofacial defect. There is an influence of domestic exposure to polycyclic aromatic hydrocarbons on women's periconceptional stage and risk of oral cleft in offspring.

Kinase activity profiling identifies putative downstream targets of cGMP/PKG signaling in inherited retinal neurodegeneration.

Inherited retinal diseases (IRDs) are a group of neurodegenerative disorders that lead to photoreceptor cell death and eventually blindness. IRDs are characterised by a high genetic heterogeneity, making it imperative to design mutation-independent therapies. Mutations in a number of IRD disease genes have been associated with a rise of cyclic 3',5'-guanosine monophosphate (cGMP) levels in photoreceptors. Accordingly, the cGMP-dependent protein kinase (PKG) has emerged as a new potential target for the mutation-independent treatment of IRDs. However, the substrates of PKG and the downstream degenerative pathways triggered by its activity have yet to be determined. Here, we performed kinome activity profiling of different murine organotypic retinal explant cultures (diseased rd1 and wild-type controls) using multiplex peptide microarrays to identify proteins whose phosphorylation was significantly altered by PKG activity. In addition, we tested the downstream effect of a known PKG inhibitor CN03 in these organotypic retina cultures. Among the PKG substrates were potassium channels belonging to the Kv1 family (KCNA3, KCNA6), cyclic AMP-responsive element-binding protein 1 (CREB1), DNA topoisomerase 2-α (TOP2A), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (F263), and the glutamate ionotropic receptor kainate 2 (GRIK2). The retinal expression of these PKG targets was further confirmed by immunofluorescence and could be assigned to various neuronal cell types, including photoreceptors, horizontal cells, and ganglion cells. Taken together, this study confirmed the key role of PKG in photoreceptor cell death and identified new downstream targets of cGMP/PKG signalling that will improve the understanding of the degenerative mechanisms underlying IRDs.

Technological advancements to study cellular signaling pathways in inherited retinal degenerative diseases.

Inherited retinal degenerative diseases (IRDs) are rare neurodegenerative disorders with mutations in hundreds of genes leading to vision loss, primarily owing to photoreceptor cell death. This genetic diversity is impeding development of effective treatment options. Gene-based therapies have resulted in the first FDA-approved drug (Luxturna) for RPE65-specific IRD. Although currently explored in clinical trials, genomic medicines are mutation-dependent, hence suitable only for patients harboring a specific mutation. Better understanding of the pathways leading to photoreceptor degeneration may help to determine common targets and develop mutation-independent therapies for larger groups of patients with IRDs. In this review, we discuss the key pathways involved in photoreceptor cell death studied by transcriptomics, proteomics, and metabolomics techniques to identify potential therapeutic targets in IRDs.

Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration.

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3',5'-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3',5'-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.