Environmental enrichment extends photoreceptor survival and visual function in a mouse model of retinitis pigmentosa

Slow, progressive rod degeneration followed by cone death leading to blindness is the pathological signature of all forms of human retinitis pigmentosa (RP). Therapeutic schemes based on intraocular delivery of neuroprotective agents prolong the lifetime of photoreceptors and have reached the stage of clinical trial. The success of these approaches depends upon optimization of chronic supply and appropriate combination of factors. Environmental enrichment (EE), a novel neuroprotective strategy based on enhanced motor, sensory and social stimulation, has already been shown to exert beneficial effects in animal models of various disorders of the CNS, including Alzheimer and Huntington disease. Here we report the results of prolonged exposure of rd10 mice, a mutant strain undergoing progressive photoreceptor degeneration mimicking human RP, to such an enriched environment from birth. By means of microscopy of retinal tissue, electrophysiological recordings, visual behaviour assessment and molecular analysis, we show that EE considerably preserves retinal morphology and physiology as well as visual perception over time in rd10 mutant mice. We find that protective effects of EE are accompanied by increased expression of retinal mRNAs for CNTF and mTOR, both factors known as instrumental to photoreceptor survival. Compared to other rescue approaches used in similar animal models, EE is highly effective, minimally invasive and results into a long-lasting retinal protection. These results open novel perspectives of research pointing to environmental strategies as useful tools to extend photoreceptor survival

The bacterial toxin CNF1 as a tool to induce retinal degeneration reminiscent of retinitis pigmentosa.

Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating still unexplored molecular pathways potentially leading to the death of these cells in inherited forms of retinal degeneratio

Site-specific abnormalities in the visual system of a mouse model of CDKL5 deficiency disorder

CDKL5 deficiency disorder (CDD) is a neurodevelopmental disorder characterized by a severe global developmental delay and early-onset seizures. Notably, patients show distinctive visual abnormalities often clinically diagnosed as cortical visual impairment. However, the involvement of cerebral cortical dysfunctions in the origin of the symptoms is poorly understood. CDD mouse models also display visual deficits, and cortical visual responses can be used as a robust biomarker in CDKL5 mutant mice. A deeper understanding of the circuits underlying the described visual deficits is essential for directing preclinical research and translational approaches. Here, we addressed this question in two ways: first, we performed an in-depth morphological analysis of the visual pathway, from the retina to the primary visual cortex (V1), of CDKL5 null mice. We found that the lack of CDKL5 produced no alteration in the organization of retinal circuits. Conversely, CDKL5 mutants showed reduced density and altered morphology of spines and decreased excitatory synapse marker PSD95 in the dorsal lateral geniculate nucleus and in V1. An increase in the inhibitory marker VGAT was selectively present in V1. Second, using a conditional CDKL5 knockout model, we showed that selective cortical deletion of CDKL5 from excitatory cells is sufficient to produce abnormalities of visual cortical responses, demonstrating that the normal function of cortical circuits is dependent on CDKL5. Intriguingly, these deficits were associated with morphological alterations of V1 excitatory and inhibitory synaptic contacts. In summary, this work proposes cortical circuit structure and function as a critically important target for studying CDD

Myriocin Effect on Tvrm4 Retina, an Autosomal Dominant Pattern of Retinitis Pigmentosa

Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary pre-requisites for translation into human therapy to slow down RP

Comparison of 5-year progression of retinitis pigmentosa involving the posterior pole among siblings by means of SD-OCT: a retrospective study

The blockchain technology promises to transform finance, money and evengovernments. However, analyses of blockchain applicability and robustness typicallyfocus on isolated systems whose actors contribute mainly by running the consensusalgorithm. Here, we highlight the importance of considering trustless platformswithin the broader ecosystem that includes social and communication networks. Asan example, we analyse the flash-crash observed on 21st June 2017 in the Ethereumplatform and show that a major phenomenon of social coordination led to acatastrophic cascade of events across several interconnected systems. We proposethe concept of “emergent centralisation” to describe situations where a single systembecomes critically important for the functioning of the whole ecosystem, and arguethat such situations are likely to become more and more frequent in interconnectedsocio-technical systems. We anticipate that the systemic approach we propose willhave implications for future assessments of trustless systems and call for the attentionof policy-makers on the fragility of our interconnected and rapidly changing world

Presynaptic partner selection during retinal circuit reassembly varies with timing of neuronal regeneration in vivo

Whether neurons can restore their original connectivity patterns during circuit repair is unclear. Taking advantage of the regenerative capacity of zebrafish retina, we show here the remarkable specificity by which surviving neurons reassemble their connectivity upon regeneration of their major input. H3 horizontal cells (HCs) normally avoid red and green cones, and prefer ultraviolet over blue cones. Upon ablation of the major (ultraviolet) input, H3 HCs do not immediately increase connectivity with other cone types. Instead, H3 dendrites retract and re-extend to contact new ultraviolet cones. But, if regeneration is delayed or absent, blue-cone synaptogenesis increases and ectopic synapses are made with red and green cones. Thus, cues directing synapse specificity can be maintained following input loss, but only within a limited time period. Further, we postulate that signals from the major input that shape the H3 HC's wiring pattern during development persist to restrict miswiring after damage

Pharmacokinetic of myriocin in rabbit’s eyes

Myriocin (Myr) is a suicide inactivator of ceramide synthesis with a complex lipid multifunctional structure. Its biological activity is exerted at very low doses, and thus highly performing quantitative method are needed [1]. The pharmacological development of Myr to modulate ceramide levels also requires currently unavailable ADME information in healthy and pathological animal models

Determination of the serine palmitoyl transferase inhibitor Myriocin by electrospray and Q-trap mass spectrometry

Myriocin, is a potent inhibitor of serine-palmitoyl-transferase, the \ufb01rst and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure Myriocin in minute specimens of animal tissue. The chemical analog 14-OH-Myriocin is used as the internal standard. The two molecules are extracted from the tissue homogenate by solid-phase extraction, separated by gradient reverse-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection is accomplished by Multiple Reaction Monitoring, employing the most representative transitions: 400@104 and 402@104 for Myriocin and 14-OH-Myriocin, respectively. The typical LoD and LLoQ of the optimized method are 0.9 pmoles/mL (approx. 0.016 pmoles injected) and 2.3 pmoles/mL, respectively, and the method is linear up to 250 pmoles/mL range (r2= 0.9996). The intra-and between-day repeatability affords a CV% 64 7.0. Applications included quantification of Myriocin in mouse lungs after 24 hrs from administration of ~4 nmoles by intra-trachea delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n=4) to 11.7 (median; 7.6-22.7 IQR, n=6) pmoles/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (<LoD) to 0.35 pmoles/retina

Novel ophthalmic formulation of myriocin: implications in retinitis pigmentosa

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP

Advancing clinical trials for inherited retinal diseases: Recommendations from the second monaciano symposium

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety