Mapping mRNA Expression of Glaucoma Genes in the Healthy Mouse Eye

Purpose/Aim: Many genes have been associated with primary open-angle glaucoma (POAG). Knowing exactly where they are expressed in the eye helps to unravel POAG pathology and to select optimal targets for intervention. We investigated whether RNA in situ hybridization (RNA-ISH) is a convenient technique to obtain detailed pan-ocular expression data of these genes. We tested this for four diverse candidate POAG genes, selected because of unclear ocular distribution (F5 and Dusp1) and relevance for potential new therapies (Tnf, Tgfβr3). Optn, a POAG gene with well-known ocular expression pattern served as control. Methods: We made a list of candidate glaucoma genes reported in genetic studies. A table of their ocular expression at the tissue level was compiled using publicly available microarray data (the ocular tissue database). To add cellular detail we performed RNA-ISH for Optn, Tnf, Tgfβr3, F5, and Dusp1 on eyes of healthy, 2-month-old, pigmented, and albino mice. Results: Expression of the Optn control matched with published immunohistochemistry data. Ocular expression of Tnf was generally low, with patches of higher Tnf expression, superficially in the corneal epithelium. F5 had a restricted expression pattern with high expression in the nonpigmented ciliary body epithelium and moderate expression in the peripapillary region. Tgfβr3 and Dusp1 showed ubiquitous expression. Conclusions: RNA-ISH is a suitable technique to determine the ocular expression pattern of POAG genes, adding meaningful cellular detail to existing microarray expression data. For instance, the high expression of F5 in the nonpigmented ciliary body epithelium suggests a role of this gene in aqueous humor dynamics and intraocular pressure. In addition, the ubiquitous expression of Tgfβr3 has implications for designing TGF-β-related glaucoma therapies, with respect to side effects. Creating pan-ocular expression maps of POAG genes with RNA-ISH will help to identify POAG pathways in speci

‘Stand still … , and move on’, a new early intervention service for cardiac arrest survivors and their caregivers: rationale and description of the intervention

This series of articles for rehabilitation in practice aims to cover a knowledge element of the rehabilitation medicine curriculum. Nevertheless they are intended to be of interest to a multidisciplinary audience. The competency addressed in this article is ‘The trainee demonstrates a knowledge of diagnostic approaches for specific impairments including cognitive dysfunction as a result of cardiac arrest.

The aqueous humor proteome of primary open angle glaucoma: An extensive review

Background: We reviewed the literature on the aqueous humor (AH) proteome of primary open angle glaucoma (POAG) patients in order to obtain deeper insight into the pathophysiology of POAG. Methods: We searched Pubmed and Embase up to May 2019 for studies that compared AH protein composition between POAG (cases) and cataract (controls). Untargeted studies (measuring the whole proteome, by LC-MS/MS) were divided into two subgroups depending on the type of surgery during which POAG AH was collected: glaucoma filtration surgery (subgroup 1) or cataract surgery (subgroup 2). We reanalyzed the raw data (subgroup 1) or combined the reported data (subgroup 2) to perform GO enrichment (GOrilla) and pathway analysis (Pathvisio). Results: Out of 93 eligible proteomic studies, seven were untargeted studies that identified 863 AH proteins. We observed 73 differentially expressed proteins in subgroup 1 and 87 differentially expressed proteins in subgroup 2. Both subgroups were characterized by activation of the acute immune response, dysregulation of folate metabolism and dysregulation of the selenium micronutrient network. For subgroup 1 but not for subgroup 2, proteins of the complement system were significantly enriched. Conclusion: AH proteome of POAG patients shows strong activation of the immune system. In addition, analysis suggests dysregulation of folate metabolism and dysregulation of selenium as underlying contributors. In view of their glaucoma surgery, POAG patients of subgroup 1 most likely are progressive whereas POAG patients in subgroup 2 most likely have stable POAG. The proteome difference between these subgroups suggests that the complement system plays a role in POAG progression

Impaired Genome Maintenance Suppresses the Growth Hormone–Insulin-Like Growth Factor 1 Axis in Mice with Cockayne Syndrome

Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb(m/m)/Xpa(−/−) mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb(m/m)/Xpa(−/−) mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb(m/m)/Xpa(−/−) and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair–deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis

UVSSA and USP7, a new couple in transcription-coupled DNA repair

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS

Age-Related Neuronal Degeneration: Complementary Roles of Nucleotide Excision Repair and Transcription-Coupled Repair in Preventing Neuropathology

Neuronal degeneration is a hallmark of many DNA repair syndromes. Yet, how DNA damage causes neuronal degeneration and whether defects in different repair systems affect the brain differently is largely unknown. Here, we performed a systematic detailed analysis of neurodegenerative changes in mouse models deficient in nucleotide excision repair (NER) and transcription-coupled repair (TCR), two partially overlapping DNA repair systems that remove helix-distorting and transcription-blocking lesions, respectively, and that are associated with the UV-sensitive syndromes xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TCR–deficient Csa−/− and Csb−/− CS mice showed activated microglia cells surrounding oligodendrocytes in regions with myelinated axons throughout the nervous system. This white matter microglia activation was not observed in NER–deficient Xpa−/− and Xpc−/− XP mice, but also occurred in XpdXPCS mice carrying a point mutation (G602D) in the Xpd gene that is associated with a combined XPCS disorder and causes a partial NER and TCR defect. The white matter abnormalities in TCR–deficient mice are compatible with focal dysmyelination in CS patients. Both TCR–deficient and NER–deficient mice showed no evidence for neuronal degeneration apart from p53 activation in sporadic (Csa−/−, Csb−/−) or highly sporadic (Xpa−/−, Xpc−/−) neurons and astrocytes. To examine to what extent overlap occurs between both repair systems, we generated TCR–deficient mice with selective inactivation of NER in postnatal neurons. These mice develop dramatic age-related cumulative neuronal loss indicating DNA damage substrate overlap and synergism between TCR and NER pathways in neurons, and they uncover the occurrence of spontaneous DNA injury that may trigger neuronal degeneration. We propose that, while Csa−/− and Csb−/− TCR–deficient mice represent powerful animal models to study the mechanisms underlying myelin abnormalities in CS, neuron-specific inactivation of NER in TCR–deficient mice represents a valuable model for the role of NER in neuronal maintenance and survival

How baseline, new-onset, and persistent depressive symptoms are associated with cardiovascular and non-cardiovascular mortality in incident patients on chronic dialysis

AbstractObjectiveDepressive symptoms are associated with mortality among patients on chronic dialysis therapy. It is currently unknown how different courses of depressive symptoms are associated with both cardiovascular and non-cardiovascular mortality.MethodsIn a Dutch prospective nation-wide cohort study among incident patients on chronic dialysis, 1077 patients completed the Mental Health Inventory, both at 3 and 12months after starting dialysis. Cox regression models were used to calculate crude and adjusted hazard ratios (HRs) for mortality for patients with depressive symptoms at 3months only (baseline only), at 12months only (new-onset), and both at 3 and 12months (persistent), using patients without depressive symptoms at 3 and 12months as reference group.ResultsDepressive symptoms at baseline only seemed to be a strong marker for non-cardiovascular mortality (HRadj 1.91, 95% CI 1.26–2.90), whereas cardiovascular mortality was only moderately increased (HRadj 1.41, 95% CI 0.85–2.33). In contrast, new-onset depressive symptoms were moderately associated with both cardiovascular (HRadj 1.66, 95% CI 1.06–2.58) and non-cardiovascular mortality (HRadj 1.46, 95% CI 0.97–2.20). Among patients with persistent depressive symptoms, a poor survival was observed due to both cardiovascular (HRadj 2.14, 95% CI 1.42–3.24) and non-cardiovascular related mortality (HRadj 1.76, 95% CI 1.20–2.59).ConclusionThis study showed that different courses of depressive symptoms were associated with a poor survival after the start of dialysis. In particular, temporary depressive symptoms at the start of dialysis may be a strong marker for non-cardiovascular mortality, whereas persistent depressive symptoms were associated with both cardiovascular and non-cardiovascular mortality

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43