Ezrin-mediated cell clearance: A new therapeutic target for adRP

Retinitis Pigmentosa (RP) is a genetic neurodegenerative disorder that causes photoreceptors cell death. Mutations in the Rhodopsin gene are common causes of autosomal dominant retinitis pigmentosa (adRP)and there is currently no available cure. Recent advances have pointed out the role of autophagy therapeutic modulation for treating genetic disorders characterized by mistrafficking and accumulation of mutated and unfolded proteins. In this regard, I recently identified Ezrin as an autophagic inhibitor in the retina. The goal of my thesis is to shed light on the mechanisms regulating cell clearance function in the RPE/retina crosstalk and to translate these discoveries into novel therapeutic strategies for adRP. I focused my effort in demonstrating the effect of Ezrin’s inhibition on clearance of misfolded RHOP23H aggregates, in vitro and in vivo. I demonstrated that pharmacological inhibition of Ezrin (NSC668394) promotes an increase of lysosomal degradation of disease-linked RHOP23H aggregates in vitro. Moreover, I demonstrated that therapeutic inhibition of Ezrin is able to decrease ER stress and to recover retina function in RHOP23H knock-in (RHOP23H/+) mice. Remarkably, treatment of RHOP23H/+ mice with NSC668394 resulted in a marked preservation of photoreceptor cells, robust decrease of cell death, culminating in an amelioration of the visual function. Finally, I gained insights into how Ezrin controls autophagy pathway and allow cellular clearance. In particular, I demonstrated that Ezrin coordinates and integrates the signal transduction pathways orchestrated by AKT/TSC with mTOR pathway to control autophagy. These findings open a new therapeutic perspective in which repression of the Ezrin may promote cell clearance reducing pathological accumulation of unfolded rhodopsin protein and rescues photoreceptor degeneration in several adRP models

MiR-211 is essential for adult cone photoreceptor maintenance and visual function.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that play an important role in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptor maintenance and function. MiR-211 is one of the most abundant miRNAs in the developing and adult eye. However, its role in controlling vertebrate visual system development, maintenance and function so far remain incompletely unexplored. Here, by targeted inactivation in a mouse model, we identify a critical role of miR-211 in cone photoreceptor function and survival. MiR-211 knockout (-/-) mice exhibited a progressive cone dystrophy accompanied by significant alterations in visual function. Transcriptome analysis of the retina from miR-211-/- mice during cone degeneration revealed significant alteration of pathways related to cell metabolism. Collectively, this study highlights for the first time the impact of miR-211 function in the retina and significantly contributes to unravelling the role of specific miRNAs in cone photoreceptor function and survival

Autophagy in the retinal pigment epithelium: a new vision and future challenges

The retinal pigment epithelium (RPE) is a highly specialised monolayer of polarized, pigmented epithelial cells that resides between the vessels of the choriocapillaris and the neural retina. The RPE is essential for the maintenance and survival of overlying light-sensitive photoreceptors, as it participates in the formation of the outer blood retinal barrier, phagocytosis, degradation of photoreceptor outer segment (POS) tips, maintenance of the retinoid cycle, and protection against light and oxidative stress. Autophagy is an evolutionarily conserved "self-eating" process, designed to maintain cellular homeostasis. The daily autophagy demands in the RPE require precise gene regulation for the digestion and recycling of intracellular and POS components in lysosomes in response to light and stress conditions. In this review, we discuss selective autophagy and focus on the recent advances in our understanding of the mechanism of cell clearance in the RPE for visual function. Understanding how this catabolic process is regulated by both transcriptional and post-transcriptional mechanisms in the RPE will promote the recognition of pathological pathways in genetic disease and shed light on potential therapeutic strategies to treat visual impairments in patients with retinal disorders associated with lysosomal dysfunction

Control of Wnt/β-Catenin Signaling Pathway in Vivo via Light Responsive Capsules

The possibility to remotely manipulate intracellular pathways in single cells is among the current goals of biomedicine, demanding new strategies to control cell function and reprogramming cell fate upon external triggering. Optogenetics is one approach in this direction, allowing specific cell stimulation by external illumination. Here, we developed optical switchers of an ancient and highly conserved system controlling a variety of developmental and adult processes in all metazoans, from Hydra to mammals, the Wnt/β-catenin signaling pathway. An intracellular modulator of the Wnt pathway was enclosed into polyelectrolyte multilayer microcapsules engineered to include self-tracking (i.e., fluorescence labeling) and light mediated heating functionalities (i.e., plasmonic nanoparticles). Capsules were delivered in vivo to Hydra and NIR triggered drug release caused forced activation of the Wnt pathway. The possibility to remotely manipulate the Wnt pathway by optical switchers may be broadly translated to achieve spatiotemporal control of cell fate for new therapeutic strategies. © 2016 American Chemical Society

Pomegranate Peel Extract as an Inhibitor of SARS-CoV-2 Spike Binding to Human ACE2 Receptor (in vitro): A Promising Source of Novel Antiviral Drugs

Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes, and triterpenes, which potentially have antiviral activities. As a consequence of the coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, thousands of scientists have been working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal of discovering effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. Using in vitro approaches, we investigated the role of a pomegranate peel extract (PPE) in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human angiotensin-converting enzyme 2 receptor, and on the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results opened new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2

Induction of Autophagy Promotes Clearance of RHOP23H Aggregates and Protects From Retinal Degeneration

Autophagy is a critical metabolic process that acts as a major self-digestion and recycling pathway contributing to maintain cellular homeostasis. An emerging field of research supports the therapeutic modulation of autophagy for treating human neurodegenerative disorders, in which toxic aggregates are accumulated in neurons. Our previous study identified Ezrin protein as an inhibitor of autophagy and lysosomal functions in the retina; thus, in turn, identifying it as a potential pharmacological target for increasing retinal cell clearance to treat inherited retinal dystrophies in which misfolded proteins have accumulated. This study aimed to verify the therapeutic inhibition of Ezrin to induce clearance of toxic aggregates in a mouse model for a dominant form of retinitis pigmentosa (i.e., RHOP23H/+). We found that daily inhibition of Ezrin significantly decreased the accumulation of misfolded RHOP23H aggregates. Remarkably, induction of autophagy, by a drug-mediated pulsatile inhibition of Ezrin, promoted the lysosomal clearance of disease-linked RHOP23H aggregates. This was accompanied with a reduction of endoplasmic reticulum (ER)-stress, robust decrease of photoreceptors' cell death, amelioration in both retinal morphology and function culminating in a better preservation of vision. Our study opens new perspectives for a pulsatile pharmacological induction of autophagy as a mutation-independent therapy paving the way toward a more effective therapeutic strategy to treat these devastating retinal disorders due to an accumulation of intracellular toxic aggregates

Retinal Degeneration in MPS-IIIA Mouse Model

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development

Pathogenic variants in IMPG1 cause autosomal dominant and autosomal recessive retinitis pigmentosa

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Real time dynamics of β-catenin expression during hydra development, regeneration and wnt signalling activation

Understanding the dynamic cellular behaviours driving morphogenesis and regeneration is a long-standing challenge in biology. Live imaging, together with genetically encoded reporters, may provide the necessary tool to address this issue, permitting the in vivo monitoring of the spatial and temporal expression dynamics of a gene of interest during a variety of developmental processes. Canonical Wnt/β-catenin signalling controls a plethora of cellular activities during development, regeneration and adulthood throughout the animal kingdom. Several reporters have been produced in animal models to reveal sites of active Wnt signalling. In order to monitor in vivo Wnt/β-catenin signalling activity in the freshwater polyp Hydra vulgaris, we generated a β-cat-eGFP transgenic Hydra, in which eGFP is driven by the Hydra β-catenin promoter.We characterized the expression dynamics during budding, regeneration and chemical activation of the Wnt/β-cat signalling pathway using light sheet fluorescence microscopy. Live imaging of the β-cat-eGFP lines recapitulated the previously reported endogenous expression pattern of β-catenin and revealed the dynamic appearance of novel sites of Wnt/β-catenin signalling, that earlier evaded detection by mean of in situ hybridization. By combining the Wnt activity read-out efficiency of the β-catenin promoter with advanced imaging, we have created a novel model system to monitor in real time the activity of Hydra β-cat regulatory sequences in vivo, and open the path to reveal β-catenin modulation in many other physiological contexts