E3 ubiquitin ligase-mediated regulation of vertebrate ocular development; new insights into the function of SIAH enzymes.

Developmental regulation of the vertebrate visual system has been a focus of investigation for generations as understanding this critical time period has direct implications on our understanding of congenital blinding disease. The majority of studies to date have focused on transcriptional regulation mediated by morphogen gradients and signaling pathways. However, recent studies of post translational regulation during ocular development have shed light on the role of the ubiquitin proteasome system (UPS). This rather ubiquitous yet highly diverse system is well known for regulating protein function and localization as well as stability via targeting for degradation by the 26S proteasome. Work from many model organisms has recently identified UPS activity during various milestones of ocular development including retinal morphogenesis, retinal ganglion cell function as well as photoreceptor homeostasis. In particular work from flies and zebrafish has highlighted the role of the E3 ligase enzyme family, Seven in Absentia Homologue (Siah) during these events. In this review, we summarize the current understanding of UPS activity during Drosophila and vertebrate ocular development, with a major focus on recent findings correlating Siah E3 ligase activity with two major developmental stages of vertebrate ocular development, retinal morphogenesis and photoreceptor specification and survival.

Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish.

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3-5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS.

Temporal characterization of optic fissure basement membrane composition suggests nidogen may be an initial target of remodeling.

Fusion of the optic fissure is necessary to complete retinal morphogenesis and ensure proper function of the optic stalk. Failure of this event leads to congenital coloboma, one of the leading causes of pediatric blindness. Mechanistically it is widely accepted that the basement membrane (BM) surrounding the maturing retina needs to be remodeled within the fissure in order to facilitate subsequent epithelial sheet fusion. However, the mechanism driving BM remodeling has yet to be elucidated. As a first step to understanding this critical molecular event we comprehensively characterized the core composition of optic fissure BMs in the zebrafish embryos. Zebrafish optic fissure BMs were found to express laminin a1, a4, b1a, c1 and c3, nidogen 1a, 1b and 2a, collagen IV a1 and a2 as well as perlecan. Furthermore, we observed that laminin, perlecan and collagen IV expression persists in the fissure during fusion, up to 56 hpf, while nidogen expression is downregulated upon initiation of fusion, at 36 hpf. Using immunohistochemistry we also show that nidogen is removed from the BM prior to that of laminin, indicating that remodeling of the BM is an ordered event. Lastly, we characterized retinal morphogenesis in the absence of nidogen function and documented retinal malformation similar to what is observed in laminin mutants. Taken together, we propose a model of BM remodeling where nidogen acts as a linchpin during initiation of optic fissure fusion.

Nitric oxide synthase inhibition impairs muscle regrowth following immobilization.

Nitric oxide (NO) has been shown to increase skeletal muscle protein synthesis. However, the role of NO during skeletal muscle regrowth after immobilization remains unknown. The purpose of this study was to determine whether NO is required for muscle regrowth/recovery after a period of disuse by immobilization. Male Wistar rats were divided into 4 groups: recovered, 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg·kg body mass-1·day-1), NG-nitro-l-arginine methyl ester (l-NAME; 90 mg·kg body mass-1·day-1), and control. The recovered, TRIM, l-NAME groups were submitted to a 7-d muscle recovery period (by remobilization), following a 10-d immobilization period (to induce plantaris [PLA] muscle atrophy). After the experimental period, the PLA muscle was collected for morphometrical (muscle fibers cross-sectional area [CSA]) and molecular (Phospho-mTORSer2448 protein expression) analysis. After 7 d of recovery, the recovered group displayed complete muscle regrowth (CSA, recovered: 2.216 ± 214 vs. CONTROL: 2.219 ± 280 cm2; P > 0.05). However, CSA of the l-NAME (1.911 ± 267 cm2) and TRIM (1.896 ± 219 cm2) groups were statistically (P < 0.05) lower than the recovered and control groups. Additionally, there was a 29% increase in Phos-mTORSer2448 protein expression levels in the recovered group compared to control group, and this increase was blocked in both TRIM and l-NAME groups. In conclusion, our results indicate that NO is crucial for skeletal muscle regrowth after an immobilization period, potentially via the mTOR signaling pathway.

Aerobic training attenuates nicotinic acethylcholine receptor changes in the diaphragm muscle during heart failure.

INTRODUCTION: Heart failure (HF) is a progressive myopathy, with clinical signs of fatigue and limb weakness that can damage the nerve-muscle interaction, altering synaptic transmission and nicotinic acetylcholine receptors (nAChR) in neuromuscular junctions (NMJs). The diaphragm is composed of a mixed proportion of muscle fibres, and during HF, this muscle becomes slower and can alter its function. As exercise training is an accepted practice to minimise abnormalities of skeletal muscle during HF, in this study, we evaluated the hypothesis that aerobic training attenuates alterations in the expression of nAChR subunits in NMJs diaphragm during heart failure. OBJECTIVE: The aim of this study was to evaluate the distribution and expression of nAChR subunits in the diaphragm muscle fibres of rats subjected to an aerobic training programme during HF. METHODS: Control (Sham), control training (ShamTR), aortic stenosis (AS) and aortic stenosis training (ASTR) groups were evaluated. The expression of nAChR subunits (γ, α1, ε, ß1 and δ) was determined by qRT-PCR, and NMJs were analysed using confocal microscopy. RESULTS: We observed increased expression of the γ, α1 and ß1 subunits in the AS group compared with the ASTR group. The distribution of NMJs was modulated in these groups. DISCUSSION: HF alters the mRNA expression of nAChR subunits and the structural characteristics of diaphragm NMJs. In addition, aerobic training did not alter NMJs morphology but attenuated the alterations in heart structure and function and in nAChR subunit mRNA expression. Our findings demonstrate the beneficial effects of aerobic exercise training in maintaining the integrity of the neuromuscular system in the diaphragm muscle during HF and may be critical for non-pharmacological therapy to improve the quality of life for patients with this syndrome.

Resistance training with excessive training load and insufficient recovery alters skeletal muscle mass-related protein expression.

The aim of this study was to investigate the effects of a resistance training program with excessive training load and insufficient recovery time between bouts on muscle hypertrophy- and atrophy-related protein expression. Male Wistar rats were randomly assigned to either a trained (TR, N = 9) or a sedentary (SE, N = 9) group. The TR group was subjected to a 12-week resistance training program with excessive training load and insufficient recovery between bouts that was designed to induce plantaris muscle atrophy. After the 12-week experiment, the plantaris muscle was collected to analyze the cross-sectional area (CSA) of the muscle fibers, and MAFbx, MyoD, myogenin, and IGF-I protein expression (Western blot). The CSA was reduced significantly (-17%, p ≤ 0.05) in the TR group compared with the SE group. Reciprocally, there was a significant (p ≤ 0.05) 20% increase in MAFbx protein expression, whereas the MyoD (-27%), myogenin (-29%), and IGF-I (-43%) protein levels decreased significantly (p ≤ 0.05) in the TR group compared with the SE group. In conclusion, our data indicated that muscle atrophy induced by resistance training with excessive training load and insufficient recovery was associated with upregulation of the MAFbx catabolic protein and downregulation of the MyoD, myogenin, and IGF-I anabolic proteins. These findings suggest that quantitative analysis of these proteins can be important and complementary with other biochemical markers to confirm a possible overtraining diagnosis.