Restoring retinal polyunsaturated fatty acid balance and retina function by targeting ceramide in AdipoR1-deficient mice.

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration. This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 fatty acids, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium. Our lipidomic and proteomic analyses indicate changes that could affect membrane composition and viscosity through altered ω3 PUFA transport and synthesis, suggesting a potential influence of AdipoR1 on these properties. Furthermore, we noted a reduction in the Bardet-Biedl syndrome proteins, which are crucial for forming and maintaining photoreceptor outer segments that are PUFA-enriched ciliary structures. Diminution in Bardet-Biedl syndrome-proteins content combined with our electron microscopic observations raises the possibility that AdipoR1 deficiency might impair ciliary function. Treatment with inhibitors of ceramide synthesis led to substantial elevation of ω3 LC-PUFAs, alleviating photoreceptor degeneration and improving retinal function. These results serve as the proof of concept for a ceramide-targeted strategy to treat retinopathies linked to PUFA deficiency, including age-related macular degeneration.

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors.

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

No evidence for magnetic field effects on the behaviour of Drosophila.

Migratory songbirds have the remarkable ability to extract directional information from the Earth's magnetic field1,2. The exact mechanism of this light-dependent magnetic compass sense, however, is not fully understood. The most promising hypothesis focuses on the quantum spin dynamics of transient radical pairs formed in cryptochrome proteins in the retina3-5. Frustratingly, much of the supporting evidence for this theory is circumstantial, largely because of the extreme challenges posed by genetic modification of wild birds. Drosophila has therefore been recruited as a model organism, and several influential reports of cryptochrome-mediated magnetic field effects on fly behaviour have been widely interpreted as support for a radical pair-based mechanism in birds6-23. Here we report the results of an extensive study testing magnetic field effects on 97,658 flies moving in a two-arm maze and on 10,960 flies performing the spontaneous escape behaviour known as negative geotaxis. Under meticulously controlled conditions and with vast sample sizes, we have been unable to find evidence for magnetically sensitive behaviour in Drosophila. Moreover, after reassessment of the statistical approaches and sample sizes used in the studies that we tried to replicate, we suggest that many-if not all-of the original results were false positives. Our findings therefore cast considerable doubt on the existence of magnetic sensing in Drosophila and thus strongly suggest that night-migratory songbirds remain the organism of choice for elucidating the mechanism of light-dependent magnetoreception.

Tuning the Metabolic Stability of Visual Cycle Modulators through Modification of an RPE65 Recognition Motif.

In the eye, the isomerization of all-trans-retinal to 11-cis-retinal is accomplished by a metabolic pathway termed the visual cycle that is critical for vision. RPE65 is the essential trans-cis isomerase of this pathway. Emixustat, a retinoid-mimetic RPE65 inhibitor, was developed as a therapeutic visual cycle modulator and used for the treatment of retinopathies. However, pharmacokinetic liabilities limit its further development including: (1) metabolic deamination of the γ-amino-α-aryl alcohol, which mediates targeted RPE65 inhibition, and (2) unwanted long-lasting RPE65 inhibition. We sought to address these issues by more broadly defining the structure-activity relationships of the RPE65 recognition motif via the synthesis of a family of novel derivatives, which were tested in vitro and in vivo for RPE65 inhibition. We identified a potent secondary amine derivative with resistance to deamination and preserved RPE65 inhibitory activity. Our data provide insights into activity-preserving modifications of the emixustat molecule that can be employed to tune its pharmacological properties.

Method for siRNA delivery in retina explants.

Several barriers prevent the delivery of nucleic acids to the retina and limit the application of established technologies, such as RNA interference (RNAi), in the study of retinae biology. Organotypic culture of retinal explants is a convenient method to decrease the complexity of the biological environment surrounding the retina while preserving most of its physiological features. Nevertheless, eliciting significant, non-toxic RNAi in retina explants is not straightforward. Retina explants are mainly constituted by neurons organized in discrete circuits embedded within a complex 3D extracellular matrix. About 70% of these neurons are post-mitotic ciliated cells that respond to light. Unfortunately, like the other cells of the retina, photoreceptors are refractory to transfection, and a toxic delivery of nucleic acid often results in permanent cell loss. RNAi has been applied to retina explants using electroporation, viral, and non-viral vectors but with reproducible, poor gene silencing efficiency. In addition, only a few superficial cells can be transduced/transfected in adult retina explants. Therefore, viruses are often injected into the eye of embryos prior to excision of the retina. However, embryonic explants are not the best model to study most retina diseases since even if they are viable for several weeks, the pathological phenotype often appears later in development. We describe a robust and straightforward method to elicit significant RNAi in adult retina explant using Reverse Magnetofection. This transfection method offers a simple tool for non-toxic gene knockdown of specific genes in adult retina explants by using cationic magnetic nanoparticles (MNPs) to complex and deliver short interfering-RNAs (siRNA) in retina cells under the action of a magnetic field.

Broadband 75-85 MHz radiofrequency fields disrupt magnetic compass orientation in night-migratory songbirds consistent with a flavin-based radical pair magnetoreceptor.

The light-dependent magnetic compass sense of night-migratory songbirds can be disrupted by weak radiofrequency fields. This finding supports a quantum mechanical, radical-pair-based mechanism of magnetoreception as observed for isolated cryptochrome 4, a protein found in birds' retinas. The exact identity of the magnetically sensitive radicals in cryptochrome is uncertain in vivo, but their formation seems to require a bound flavin adenine dinucleotide chromophore and a chain of four tryptophan residues within the protein. Resulting from the hyperfine interactions of nuclear spins with the unpaired electrons, the sensitivity of the radicals to radiofrequency magnetic fields depends strongly on the number of magnetic nuclei (hydrogen and nitrogen atoms) they contain. Quantum-chemical calculations suggested that electromagnetic noise in the frequency range 75-85 MHz could give information about the identity of the radicals involved. Here, we show that broadband 75-85 MHz radiofrequency fields prevent a night-migratory songbird from using its magnetic compass in behavioural experiments. These results indicate that at least one of the components of the radical pair involved in the sensory process of avian magnetoreception must contain a substantial number of strong hyperfine interactions as would be the case if a flavin-tryptophan radical pair were the magnetic sensor.

Magnetically Assisted Drug Delivery of Topical Eye Drops Maintains Retinal Function In Vivo in Mice.

Barded-Biedl syndrome (BBS) is a rare genetic disorder with an unmet medical need for retinal degeneration. Small-molecule drugs were previously identified to slow down the apoptosis of photoreceptors in BBS mouse models. Clinical translation was not practical due to the necessity of repetitive invasive intravitreal injections for pediatric populations. Non-invasive methods of retinal drug targeting are a prerequisite for acceptable adaptation to the targeted pediatric patient population. Here, we present the development and functional testing of a non-invasive, topical, magnetically assisted delivery system, harnessing the ability of magnetic nanoparticles (MNPs) to cargo two drugs (guanabenz and valproic acid) with anti-unfolded protein response (UPR) properties towards the retina. Using magnetic resonance imaging (MRI), we showed the MNPs' presence in the retina of Bbs wild-type mice, and their photoreceptor localization was validated using transmission electron microscopy (TEM). Subsequent electroretinogram recordings (ERGs) demonstrated that we achieved beneficial biological effects with the magnetically assisted treatment translating the maintained light detection in Bbs-/- mice (KO). To our knowledge, this is the first demonstration of efficient magnetic drug targeting in the photoreceptors in vivo after topical administration. This non-invasive, needle-free technology expands the application of SMDs for the treatment of a vast spectrum of retinal degenerations and other ocular diseases.

Magnetic sensitivity of cryptochrome 4 from a migratory songbird.

Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds4-7. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin-tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds.

New Method for Efficient siRNA Delivery in Retina Explants: Reverse Magnetofection.

The prevalence of retinal disorders associated with visual impairment and blindness is increasing worldwide, while most of them remain without effective treatment. Pharmacological and molecular therapy development is hampered by the lack of effective drug delivery into the posterior segment of the eye. Among molecular approaches, RNA-interference (RNAi) features strong advantages, yet delivering it to the inner layer of the retina appears extremely challenging. To address this, we developed an original magnetic nanoparticles (MNPs)-based transfection method that allows the efficient delivery of siRNA in all retinal layers of rat adult retinas through magnetic targeting. To establish delivery of RNAi throughout the retina, we have chosen organotypic retinal explants as an ex vivo model and for future high content screening of molecular drugs. Conversely to classic Magnetofection, and similar to conditions in the posterior chamber of the eye, our methods allows attraction of siRNA complexed to MNPs from the culture media into the explant. Our method termed "Reverse Magnetofection" provides a novel and nontoxic strategy for RNAi-based molecular as well as gene therapy in the retina that can be transferred to a wide variety of organ explants.

Efficient Ocular Delivery of VCP siRNA via Reverse Magnetofection in RHO P23H Rodent Retina Explants.

The use of synthetic RNA for research purposes as well as RNA-based therapy and vaccination has gained increasing importance. Given the anatomical seclusion of the eye, small interfering RNA (siRNA)-induced gene silencing bears great potential for targeted reduction of pathological gene expression that may allow rational treatment of chronic eye diseases in the future. However, there is yet an unmet need for techniques providing safe and efficient siRNA delivery to the retina. We used magnetic nanoparticles (MNPs) and magnetic force (Reverse Magnetofection) to deliver siRNA/MNP complexes into retinal explant tissue, targeting valosin-containing protein (VCP) previously established as a potential therapeutic target for autosomal dominant retinitis pigmentosa (adRP). Safe and efficient delivery of VCP siRNA was achieved into all retinal cell layers of retinal explants from the RHO P23H rat, a rodent model for adRP. No toxicity or microglial activation was observed. VCP silencing led to a significant decrease of retinal degeneration. Reverse Magnetofection thus offers an effective method to deliver siRNA into retinal tissue. Used in combination with retinal organotypic explants, it can provide an efficient and reliable preclinical test platform of RNA-based therapy approaches for ocular diseases.