The role of the serotonergic system in atropine's anti-myopic effects.

The muscarinic cholinergic antagonist atropine is the most widely used pharmacological treatment for the visual disorder myopia (short-sightedness), the leading cause of low-vision worldwide. This study sought to better define the mechanism by which atropine inhibits myopic growth. Although classified as a muscarinic-cholinergic antagonist, atropine has been found to bind and modulate the activity of several non-cholinergic systems (e.g., serotonin). Thus, this study investigated whether the serotonergic system could underly atropine's anti-myopic effects. Using a chick model of myopia, we report that atropine's growth-inhibitory effects can be attenuated by pharmacological stimulation of the serotonin system. This may suggest that atropine can slow the development of myopia through inhibiting serotonergic receptor activity. We also observed that pharmacological antagonism of serotonergic receptors inhibits the development of experimental myopia in a dose-dependent manner, further demonstrating that modulation of serotonergic receptor activity can alter ocular growth rates. Finally, we found that neither experimental myopia, nor atropine treatment, induced a significant change in retinal serotonergic output (i.e., synthesis, transport, release and catabolism). This may suggest that, although myopic growth can be inhibited through modulation of serotonergic receptor activity (by atropine or serotonergic antagonists), this does not require a change in serotonin levels. These findings regarding a serotonergic mechanism for atropine may have significant ramifications for the treatment of human myopia. This includes assessing the use of atropine in patients who are also undergoing treatment to upregulate serotonergic signaling (e.g., serotonergic anti-depressants).

Rhinovirus protease cleavage of nucleoporins: perspective on implications for airway remodeling.

Human Rhinoviruses (RV) are a major cause of common colds and infections in early childhood and can lead to subsequent development of asthma via an as yet unknown mechanism. Asthma is a chronic inflammatory pulmonary disease characterized by significant airway remodeling. A key component of airway remodeling is the transdifferentiation of airway epithelial and fibroblast cells into cells with a more contractile phenotype. Interestingly, transforming growth factor-beta (TGF-ß), a well characterized inducer of transdifferentiation, is significantly higher in airways of asthmatics compared to non-asthmatics. RV infection induces TGF-ß signaling, at the same time nucleoporins (Nups), including Nup153, are cleaved by RV proteases disrupting nucleocytoplasmic transport. As Nup153 regulates nuclear export of SMAD2, a key intermediate in the TGF-ß transdifferentiation pathway, its loss of function would result in nuclear retention of SMAD2 and dysregulated TGF-ß signaling. We hypothesize that RV infection leads to increased nuclear SMAD2, resulting in sustained TGF-ß induced gene expression, priming the airway for subsequent development of asthma. Our hypothesis brings together disparate studies on RV, asthma and Nup153 with the aim to prompt new research into the role of RV infection in development of asthma.

The safety and tolerability of levodopa eye drops for the treatment of ocular disorders: A randomized first-in-human study.

Myopia is the leading cause of low vision worldwide and can lead to significant pathological complications. Therefore, to improve patient outcomes, the field continues to develop novel interventions for this visual disorder. Accordingly, this first-in-human study reports on the safety profile of a novel dopamine-based ophthalmic treatment for myopia, levodopa/carbidopa eye drops. This phase I, first-in-human, monocenter, placebo-controlled, double-blind, paired-eye, multidose, randomized clinical trial was undertaken in healthy adult males aged 18-30 years (mean age 24.9 ± 2.7) at the University of Canberra Eye Clinic, Australia. Participants were randomly assigned to receive either a low (1.4 levodopa:0.34 carbidopa [µmoles/day], n = 14) or standard dose (2.7 levodopa:0.68 carbidopa [µmoles/day], n = 15) of levodopa/carbidopa eye drops in one eye and placebo in the fellow eye once daily for 4 weeks (28 days). Over this 4-week trial, and after a 4-month follow-up visit, levodopa/carbidopa treatment had no significant effect on ocular tolerability and anterior surface integrity, visual function, ocular health, refraction/ocular biometry, and did not induce any non-ocular adverse events. These results indicate that topical levodopa/carbidopa is safe and tolerable to the eye, paving the way for future studies on the efficacy of this novel ophthalmic formulation in the treatment of human myopia. The findings of this study have implications not only for the treatment of myopia, but in a number of other visual disorders (i.e., amblyopia, diabetic retinopathy, and age-related macular degeneration) in which levodopa has been identified as a potential clinical intervention.

Correlation between small-scale methylation changes and gene expression during the development of myopia.

Visually induced changes in the expression of early growth response-1 (EGR1), FBJ osteosarcoma oncogene (FOS), and NGFI-A binding protein-2 (NAB2) appear to form a part of a retinal network fundamental to ocular growth regulation, and thus, the development of myopia (short-sightedness). However, it is unclear how environmental (visual) cues are translated into these molecular changes. One possibility is through epigenetic modifications such as DNA methylation, a known regulator of such processes. By sequencing bisulfite-converted DNA amplicons, this study examined whether changes in DNA methylation occur within specific regulatory and promoter regions of EGR1, FOS, and NAB2 during the periods of increased and decreased ocular growth in chicks. Visually induced changes in ocular growth rates were associated with single-point, but not large-scale, shifts in methylation levels within the investigated regions. Analysis of methylation pattern variability (entropy) demonstrated that the observed methylation changes are occurring within small subpopulations of retinal cells. This concurs with previous observations that EGR1 and FOS are differentially regulated at the peptide level within specific retinal cell types. Together, the findings of this study support a potential role for DNA methylation in the translation of external visual cues into molecular changes critical for ocular growth regulation and myopia development.

Transcriptome-based insights into gene networks controlling myopia prevention.

Myopia (short-sightedness), usually caused by excessive elongation of the eye during development, has reached epidemic proportions worldwide. In animal systems including the chicken model, several treatments have been shown to inhibit ocular elongation and experimental myopia. Although diverse in their apparent mechanism of action, each one leads to a reduction in the rate of ocular growth. We hypothesize that a defined set of retinal molecular changes may underlie growth inhibition, irrespective of the treatment agent used. Accordingly, across five well-established but diverse methods of inhibiting myopia, significant overlap is seen in the retinal transcriptome profile (transcript levels and alternative splicing events) in chicks when analyzed by RNA-seq. Within the two major pathway networks enriched during growth inhibition, that of cell signaling and circadian entrainment, transcription factors form the largest functional grouping. Importantly, a large percentage of those genes forming the defined retinal response are downstream targets of the transcription factor EGR1 which itself shows a universal response to all five growth-inhibitory treatments. This supports EGR1's previously implicated role in ocular growth regulation. Finally, by contrasting our data with human linkage and GWAS studies on refractive error, we confirm the applicability of our study to the human condition. Together, these findings suggest that a universal set of transcriptome changes, which sit within a well-defined retinal network that cannot be bypassed, is fundamental to growth regulation, thus paving a way for designing novel targets for myopia therapies.

Insights into the mechanism by which atropine inhibits myopia: evidence against cholinergic hyperactivity and modulation of dopamine release.

BACKGROUND AND PURPOSE: The ability of the muscarinic cholinergic antagonist atropine to inhibit myopia development in humans and animal models would suggest that cholinergic hyperactivity may underlie myopic growth. To test this, we investigated whether cholinergic agonists accelerate ocular growth rates in chickens. Furthermore, we investigated whether atropine alters ocular growth by downstream modulation of dopamine levels, a mechanism postulated to underlie its antimyopic effects. EXPERIMENTAL APPROACH: Muscarinic (muscarine and pilocarpine), nicotinic (nicotine) and non-specific (oxotremorine and carbachol) cholinergic agonists were administered to chicks developing form-deprivation myopia (FDM) or chicks that were otherwise untreated. Vitreal levels of dopamine and its primary metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were examined using mass spectrometry MS in form-deprived chicks treated with atropine (360, 15 or 0.15 nmol). Further, we investigated whether dopamine antagonists block atropine's antimyopic effects. KEY RESULTS: Unexpectedly, administration of each cholinergic agonist inhibited FDM but did not affect normal ocular development. Atropine only affected dopamine and DOPAC levels at its highest dose. Dopamine antagonists did not alter the antimyopia effects of atropine. CONCLUSION AND IMPLICATIONS: Muscarinic, nicotinic and non-specific cholinergic agonists inhibited FDM development. This indicates that cholinergic hyperactivity does not underlie myopic growth and questions whether atropine inhibits myopia via cholinergic antagonism. This study also demonstrates that changes in retinal dopamine release are not required for atropine's antimyopic effects. Finally, nicotinic agonists may represent a novel and more targeted approach for the cholinergic control of myopia as they are unlikely to cause the anterior segment side effects associated with muscarinic treatment.

Coadministration With Carbidopa Enhances the Antimyopic Effects of Levodopa in Chickens.

Purpose: Topical application of levodopa inhibits the development of form-deprivation myopia (FDM) and lens-induced myopia (LIM) in chicks. Here we examine whether coadministration with carbidopa enhances this protection and compare the effectiveness of topical versus systemic administration. We also investigate the degree to which topical and systemic administration of these compounds alters retinal dopamine release and examine whether this is the mechanism by which they inhibit experimental myopia. Methods: Levodopa and levodopa:carbidopa (at a 4:1 ratio) were administered as twice-daily eye drops or once-daily intraperitoneal injections to chicks developing FDM or LIM over an ascending dose range. Axial length and refraction were measured following 4 days of treatment. Dopamine levels in the vitreous and blood were analyzed using liquid chromatography-mass spectrometry following topical or systemic administration of levodopa or levodopa:carbidopa. Finally, chicks receiving topical or systemic levodopa or levodopa:carbidopa were cotreated with the dopamine antagonist spiperone. Results: Levodopa:carbidopa inhibited the development of FDM and LIM to a greater extent than levodopa alone (P < 0.05). Topical application was more effective than systemic administration (P < 0.001). Vitreal dopamine levels were increased to the greatest extent by topical application of levodopa:carbidopa (P < 0.001). Systemic but not topical administration significantly increased dopamine levels within the blood (P < 0.01). Cotreatment with spiperone inhibited the antimyopic effects (P < 0.05) of levodopa and levodopa:carbidopa. Conclusions: The presence of carbidopa increases the bioavailability of levodopa within the eye, enhancing its antimyopic effects, with topical application showing the greatest efficacy. Thus levodopa:carbidopa may be a promising treatment for controlling the progression of human myopia.

Conservation and turnover of miRNAs and their highly complementary targets in early branching animals.

MicroRNAs (miRNAs) are crucial post-transcriptional regulators that have been extensively studied in Bilateria, a group comprising the majority of extant animals, where more than 30 conserved miRNA families have been identified. By contrast, bilaterian miRNA targets are largely not conserved. Cnidaria is the sister group to Bilateria and thus provides a unique opportunity for comparative studies. Strikingly, like their plant counterparts, cnidarian miRNAs have been shown to predominantly have highly complementary targets leading to transcript cleavage by Argonaute proteins. Here, we assess the conservation of miRNAs and their targets by small RNA sequencing followed by miRNA target prediction in eight species of Anthozoa (sea anemones and corals), the earliest-branching cnidarian class. We uncover dozens of novel miRNAs but only a few conserved ones. Further, given their high complementarity, we were able to computationally identify miRNA targets in each species. Besides evidence for conservation of specific miRNA target sites, which are maintained between sea anemones and stony corals across 500 Myr of evolution, we also find indications for convergent evolution of target regulation by different miRNAs. Our data indicate that cnidarians have only few conserved miRNAs and corresponding targets, despite their high complementarity, suggesting a high evolutionary turnover.

Rural-urban differences in myopia prevalence among myopes presenting to Bhutanese retinal clinical services: a 3-year national study.

PURPOSE: To determine the prevalence and demographic characteristics of myopia among patients presenting to the national vitreo-retinal (VR) services in Bhutan. METHODS: The records of the VR clinic at the apex national referral centre, providing the only VR services in the country, were reviewed to identify all new myopia patients over three years. Thus, we surveyed all referrals nationally. The patients were categorised into urban and rural females and males. We assessed myopia prevalence in each group by occupational and educational categories. We examined univariate prevalence data and a multivariate logistic regression (MLR) identified independent factors. RESULTS: Of 2913 cases 1544 (53.0%) were males. Females presented earlier (mean ±SD): overall 45.7 ± 21.9 cf. 48.6 ± 21.6 years, p = 0.003, and among myopes 23.9 ± 13.5 cf. 27.6 ± 18.6 years, p = 0.032. Myopia constituted 92.1% of refractive error, an overall prevalence of 12.3%. Myopia was more common among females (p = 0.01) and urbanites (p = 0.02). Myopia prevalence was highest among urban females (20.9%), followed by urban males (11.9%), rural females (6.8%), and rural males (5.2%). Logistic regression revealed that the odds of having myopia were increased by being a student (4.96 ×) or professional (1.96 ×), and decreased by rural living (1.75 ×), all p ≤ 0.038. CONCLUSIONS: This is the first study on myopia in Bhutan. As observed throughout East and Southeast Asia, the prevalence of myopia was higher in females and urbanites and positively associated with formal education. Given known risk factors, these prevalences may be driven by educational pressures and reduced time spent outdoors.

Form-Deprivation and Lens-Induced Myopia Are Similarly Affected by Pharmacological Manipulation of the Dopaminergic System in Chicks.

Purpose: Animal models have demonstrated a link between decreases in retinal dopamine levels and the development of form-deprivation myopia (FDM). However, the consistency of dopamine's role in the other major form of experimental myopia, that of lens-induced myopia (LIM), is less clear, raising the question as to what extent dopamine plays a role in human myopia. Therefore, to better define the role of dopamine in both forms of experimental myopia, we examined how consistent the protection afforded by dopamine and the dopamine agonist 6-amino-5,6,7,8-tetrahydronaphthalene-2,3-diol hydrobromide (ADTN) is between FDM and LIM. Methods: Intravitreal injections of dopamine (0.002, 0.015, 0.150, 1.500 µmol) or ADTN (0.001, 0.010, 0.100, 1.000 µmol) were administered daily to chicks developing FDM or LIM. Axial length and refraction were measured following 4 days of treatment. To determine the receptor subtype by which dopamine and ADTN inhibit FDM and LIM, both compounds were coadministered with either the dopamine D2-like antagonist spiperone (0.005 µmol) or the D1-like antagonist SCH-23390 (0.005 µmol). Results: Intravitreal administration of dopamine or ADTN inhibited the development of FDM (ED50 = 0.003 µmol and ED50 = 0.011 µmol, respectively) and LIM (ED50 = 0.002 µmol and ED50 = 0.010 µmol, respectively) in a dose-dependent manner, with a similar degree of protection observed in both paradigms (P = 0.471 and P = 0.969, respectively). Coadministration with spiperone, but not SCH-23390, inhibited the protective effects of dopamine and ADTN against the development of both FDM (P = 0.214 and P = 0.138, respectively) and LIM (P = 0.116 and P = 0.100, respectively). Conclusions: pharmacological targeting of the retinal dopamine system inhibits FDM and LIM in a similar dose-dependent manner through a D2-like mechanism.

Topical application of dopaminergic compounds can inhibit deprivation myopia in chicks.

PURPOSE: Animal models have demonstrated a link between dysregulation of the retinal dopamine system and the development of experimental myopia (short-sightedness). However, pharmacological investigations of dopamine in animal models rely heavily on intravitreal or systemic administration, which have several limitations for longer-term experiments. We therefore investigated whether administration of dopamine as a topical eye drop can inhibit the development of form-deprivation myopia (FDM) in chicks. We also examined whether chemical modification of dopamine through deuterium substitution, which might enhance stability and bioavailability, can increase dopamine's effectiveness against FDM when given topically. METHODS: Dopamine or deuterated dopamine (Dopamine-1,1,2,2-d4 hydrochloride) was administered as a daily intravitreal injection or as daily topical eye drops to chicks developing FDM over an ascending dose range (min. n = 6 per group). Axial length and refraction were measured following 4 days of treatment. RESULTS: Both intravitreal (ED50 = 0.002µmoles) and topical application (ED50 = 6.10µmoles) of dopamine inhibited the development of FDM in a dose-dependent manner. Intravitreal injections, however, elicited a significantly higher level of protection relative to topical eye drops (p < 0.01). Deuterated dopamine inhibited FDM to a similar extent as unmodified dopamine when administered as intravitreal injections (p = 0.897) or topical eye drops (p = 0.921). CONCLUSIONS: Both intravitreal and topical application of dopamine inhibit the development of FDM in a dose-dependent manner, indicating that topical administration may be an effective avenue for longer-term dopamine experiments. Deuterium substitution does not alter the protection afforded by dopamine against FDM when given as either an intravitreal injection or topical eye drop.

Levodopa inhibits the development of lens-induced myopia in chicks.

Animal models have demonstrated a link between dysregulation of the retinal dopamine system and the development of myopia (short-sightedness). We have previously demonstrated that topical application of levodopa in chicks can inhibit the development of form-deprivation myopia (FDM) in a dose-dependent manner. Here, we examine whether this same protection is observed in lens-induced myopia (LIM), and whether levodopa's protection against FDM and LIM occurs through a dopamine D1- or D2-like receptor mechanism. To do this, levodopa was first administered daily as an intravitreal injection or topical eye drop, at one of four ascending doses, to chicks developing LIM. Levodopa's mechanism of action was then examined by co-administration of levodopa injections with D1-like (SCH-23390) or D2-like (spiperone) dopamine antagonists in chicks developing FDM or LIM. For both experiments, levodopa's effectiveness was examined by measuring axial length and refraction after 4 days of treatment. Levodopa inhibited the development of LIM in a dose-dependent manner similar to its inhibition of FDM when administered via intravitreal injections or topical eye drops. In both FDM and LIM, levodopa injections remained protective against myopia when co-administered with SCH-23390, but not spiperone, indicating that levodopa elicits its protection through a dopamine D2-like receptor mechanism in both paradigms.

Effectiveness and safety of topical levodopa in a chick model of myopia.

Animal models have demonstrated a link between dysregulation of the retinal dopamine system and the excessive ocular growth associated with the development of myopia. Here we show that intravitreal or topical application of levodopa, which is widely used in the treatment of neurological disorders involving dysregulation of the dopaminergic system, inhibits the development of experimental myopia in chickens. Levodopa slows ocular growth in a dose dependent manner in chicks with a similar potency to atropine, a common inhibitor of ocular growth in humans. Topical levodopa remains effective over chronic treatment periods, with its effectiveness enhanced by coadministration with carbidopa to prevent its premature metabolism. No changes in normal ocular development (biometry and refraction), retinal health (histology), or intraocular pressure were observed in response to chronic treatment (4 weeks). With a focus on possible clinical use in humans, translation of these avian safety findings to a mammalian model (mouse) illustrate that chronic levodopa treatment (9 months) does not induce any observable changes in visual function (electroretinogram recordings), ocular development, and retinal health, suggesting that levodopa may have potential as a therapeutic intervention for human myopia.

The epidemics of myopia: Aetiology and prevention.

There is an epidemic of myopia in East and Southeast Asia, with the prevalence of myopia in young adults around 80-90%, and an accompanying high prevalence of high myopia in young adults (10-20%). This may foreshadow an increase in low vision and blindness due to pathological myopia. These two epidemics are linked, since the increasingly early onset of myopia, combined with high progression rates, naturally generates an epidemic of high myopia, with high prevalences of "acquired" high myopia appearing around the age of 11-13. The major risk factors identified are intensive education, and limited time outdoors. The localization of the epidemic appears to be due to the high educational pressures and limited time outdoors in the region, rather than to genetically elevated sensitivity to these factors. Causality has been demonstrated in the case of time outdoors through randomized clinical trials in which increased time outdoors in schools has prevented the onset of myopia. In the case of educational pressures, evidence of causality comes from the high prevalence of myopia and high myopia in Jewish boys attending Orthodox schools in Israel compared to their sisters attending religious schools, and boys and girls attending secular schools. Combining increased time outdoors in schools, to slow the onset of myopia, with clinical methods for slowing myopic progression, should lead to the control of this epidemic, which would otherwise pose a major health challenge. Reforms to the organization of school systems to reduce intense early competition for accelerated learning pathways may also be important.

Contrasting Sex-and Caste-Dependent piRNA Profiles in the Transposon Depleted Haplodiploid Honeybee Apis mellifera.

Protecting genome integrity against transposable elements is achieved by intricate molecular mechanisms involving PIWI proteins, their associated small RNAs (piRNAs), and epigenetic modifiers such as DNA methylation. Eusocial bees, in particular the Western honeybee, Apis mellifera, have one of the lowest contents of transposable elements in the animal kingdom, and, unlike other animals with a functional DNA methylation system, appear not to methylate their transposons. This raises the question of whether the PIWI machinery has been retained in this species. Using comparative genomics, mass spectrometry, and expressional profiling, we present seminal evidence that the piRNA system is conserved in honeybees. We show that honey bee piRNAs contain a 2'-O-methyl modification at the 3' end, and have a bias towards a 5' terminal U, which are signature features of their biogenesis. Both piRNA repertoire and expression levels are greater in reproductive individuals than in sterile workers. Haploid males, where the detrimental effects of transposons are dominant, have the greatest piRNA levels, but surprisingly, the highest expression of transposons. These results show that even in a transposon-depleted species, the piRNA system is required to guard the vulnerable haploid genome and reproductive castes against transposon-associated genomic instability. This also suggests that dosage plays an important role in the regulation of transposons and piRNAs expression in haplo-diploid systems.

Developmental and loco-like effects of a swainsonine-induced inhibition of α-mannosidase in the honey bee, Apis mellifera.

BACKGROUND: Deficiencies in lysosomal a-mannosidase (LAM) activity in animals, caused either by mutations or by consuming toxic alkaloids, lead to severe phenotypic and behavioural consequences. Yet, epialleles adversely affecting LAM expression exist in the honey bee population suggesting that they might be beneficial in certain contexts and cannot be eliminated by natural selection. METHODS: We have used a combination of enzymology, molecular biology and metabolomics to characterise the catalytic properties of honey bee LAM (AmLAM) and then used an indolizidine alkaloid swainsonine to inhibit its activity in vitro and in vivo. RESULTS: We show that AmLAM is inhibited in vitro by swainsonine albeit at slightly higher concentrations than in other animals. Dietary exposure of growing larvae to swainsonine leads to pronounced metabolic changes affecting not only saccharides, but also amino acids, polyols and polyamines. Interestingly, the abundance of two fatty acids implicated in epigenetic regulation is significantly reduced in treated individuals. Additionally, swainsonie causes loco-like symptoms, increased mortality and a subtle decrease in the rate of larval growth resulting in a subsequent developmental delay in pupal metamorphosis. DISCUSSION: We consider our findings in the context of cellular LAM function, larval development, environmental toxicity and colony-level impacts. The observed developmental heterochrony in swainsonine-treated larvae with lower LAM activity offer a plausible explanation for the existence of epialleles with impaired LAM expression. Individuals carrying such epialleles provide an additional level of epigenetic diversity that could be beneficial for the functioning of a colony whereby more flexibility in timing of adult emergence might be useful for task allocation.

Animal Studies and the Mechanism of Myopia-Protection by Light?

Epidemiological studies have demonstrated that spending time outdoors during your childhood is protective against the development of myopia. It has been hypothesized that this protective effect is associated with light-induced increases in retinal dopamine levels, a critical neuromodulator that has long been postulated to be involved in the regulation of ocular growth. This paper, along with the paper entitled "What do animal studies tell us about the mechanism of myopia-protection by light?" discusses the evidence provided by animal models for this hypothesis.