The Role of Inflammation in Retinal Neurodegeneration and Degenerative Diseases

Retinal neurodegeneration is predominantly reported as the apoptosis or impaired function of the photoreceptors. Retinal degeneration is a major causative factor of irreversible vision loss leading to blindness. In recent years, retinal degenerative diseases have been investigated and many genes and genetic defects have been elucidated by many of the causative factors. An enormous amount of research has been performed to determine the pathogenesis of retinal degenerative conditions and to formulate the treatment modalities that are the critical requirements in this current scenario. Encouraging results have been obtained using gene therapy. We provide a narrative review of the various studies performed to date on the role of inflammation in human retinal degenerative diseases such as age-related macular degeneration, inherited retinal dystrophies, retinitis pigmentosa, Stargardt macular dystrophy, and Leber congenital amaurosis. In addition, we have highlighted the pivotal role of various inflammatory mechanisms in the progress of retinal degeneration. This review also offers an assessment of various therapeutic approaches, including gene-therapies and stem-cell-based therapies, for degenerative retinal diseases.     

Retinal Organoids and Retinal Prostheses: An Overview

Despite the progress of modern medicine in the last decades, millions of people diagnosed with retinal dystrophies (RDs), such as retinitis pigmentosa, or age-related diseases, such as age-related macular degeneration, are suffering from severe visual impairment or even legal blindness. On the one hand, the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and the progress of three-dimensional (3D) retinal organoids (ROs) technology provide a great opportunity to study, understand, and even treat retinal diseases. On the other hand, research advances in the field of electronic retinal prosthesis using inorganic photovoltaic polymers and the emergence of organic semiconductors represent an encouraging therapeutical strategy to restore vision to patients at the late onset of the disease. This review will provide an overview of the latest advancement in both fields. We first describe the retina and the photoreceptors, briefly mention the most used RD animal models, then focus on the latest RO differentiation protocols, carry out an overview of the current technology on inorganic and organic retinal prostheses to restore vision, and finally summarize the potential utility and applications of ROs.     

Retinal Organoid Technology: Where Are We Now?

It is difficult to regenerate mammalian retinal cells once the adult retina is damaged, and current clinical approaches to retinal damages are very limited. The introduction of the retinal organoid technique empowers researchers to study the molecular mechanisms controlling retinal development, explore the pathogenesis of retinal diseases, develop novel treatment options, and pursue cell/tissue transplantation under a certain genetic background. Here, we revisit the historical background of retinal organoid technology, categorize current methods of organoid induction, and outline the obstacles and potential solutions to next-generation retinal organoids. Meanwhile, we recapitulate recent research progress in cell/tissue transplantation to treat retinal diseases, and discuss the pros and cons of transplanting single-cell suspension versus retinal organoid sheet for cell therapies.     

Patient-Derived Organoids as a Model for Cancer Drug Discovery

In the search for the ideal model of tumours, the use of three-dimensional in vitro models is advancing rapidly. These are intended to mimic the in vivo properties of the tumours which affect cancer development, progression and drug sensitivity, and take into account cell–cell interactions, adhesion and invasiveness. Importantly, it is hoped that successful recapitulation of the structure and function of the tissue will predict patient response, permitting the development of personalized therapy in a timely manner applicable to the clinic. Furthermore, the use of co-culture systems will allow the role of the tumour microenvironment and tissue–tissue interactions to be taken into account and should lead to more accurate predictions of tumour development and responses to drugs. In this review, the relative merits and limitations of patient-derived organoids will be discussed compared to other in vitro and ex vivo cancer models. We will focus on their use as models for drug testing and personalized therapy and how these may be improved. Developments in technology will also be considered, including the use of microfluidics, 3D bioprinting, cryopreservation and circulating tumour cell-derived organoids. These have the potential to enhance the consistency, accessibility and availability of these models.     

An Update on Gene Therapy for Inherited Retinal Dystrophy: Experience in Leber Congenital Amaurosis Clinical Trials

Inherited retinal dystrophies (IRDs) are a group of rare eye diseases caused by gene mutations that result in the degradation of cone and rod photoreceptors or the retinal pigment epithelium. Retinal degradation progress is often irreversible, with clinical manifestations including color or night blindness, peripheral visual defects and subsequent vision loss. Thus, gene therapies that restore functional retinal proteins by either replenishing unmutated genes or truncating mutated genes are needed. Coincidentally, the eye’s accessibility and immune-privileged status along with major advances in gene identification and gene delivery systems heralded gene therapies for IRDs. Among these clinical trials, voretigene neparvovec-rzyl (Luxturna), an adeno-associated virus vector-based gene therapy drug, was approved by the FDA for treating patients with confirmed biallelic RPE65 mutation-associated Leber Congenital Amaurosis (LCA) in 2017. This review includes current IRD gene therapy clinical trials and further summarizes preclinical studies and therapeutic strategies for LCA, including adeno-associated virus-based gene augmentation therapy, 11-cis-retinal replacement, RNA-based antisense oligonucleotide therapy and CRISPR-Cas9 gene-editing therapy. Understanding the gene therapy development for LCA may accelerate and predict the potential hurdles of future therapeutics translation. It may also serve as the template for the research and development of treatment for other IRDs.     

Energy Metabolism in the Inner Retina in Health and Glaucoma

Glaucoma, the leading cause of irreversible blindness, is a heterogeneous group of diseases characterized by progressive loss of retinal ganglion cells (RGCs) and their axons and leads to visual loss and blindness. Risk factors for the onset and progression of glaucoma include systemic and ocular factors such as older age, lower ocular perfusion pressure, and intraocular pressure (IOP). Early signs of RGC damage comprise impairment of axonal transport, downregulation of specific genes and metabolic changes. The brain is often cited to be the highest energy-demanding tissue of the human body. The retina is estimated to have equally high demands. RGCs are particularly active in metabolism and vulnerable to energy insufficiency. Understanding the energy metabolism of the inner retina, especially of the RGCs, is pivotal for understanding glaucoma’s pathophysiology. Here we review the key contributors to the high energy demands in the retina and the distinguishing features of energy metabolism of the inner retina. The major features of glaucoma include progressive cell death of retinal ganglions and optic nerve damage. Therefore, this review focuses on the energetic budget of the retinal ganglion cells, optic nerve and the relevant cells that surround them.     

Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.     

Modeling PCDH19-CE: From 2D Stem Cell Model to 3D Brain Organoids

PCDH19 clustering epilepsy (PCDH19-CE) is a genetic disease characterized by a heterogeneous phenotypic spectrum ranging from focal epilepsy with rare seizures and normal cognitive development to severe drug-resistant epilepsy associated with intellectual disability and autism. Unfortunately, little is known about the pathogenic mechanism underlying this disease and an effective treatment is lacking. Studies with zebrafish and murine models have provided insights on the function of PCDH19 during brain development and how its altered function causes the disease, but these models fail to reproduce the human phenotype. Induced pluripotent stem cell (iPSC) technology has provided a complementary experimental approach for investigating the pathogenic mechanisms implicated in PCDH19-CE during neurogenesis and studying the pathology in a more physiological three-dimensional (3D) environment through the development of brain organoids. We report on recent progress in the development of human brain organoids with a particular focus on how this 3D model may shed light on the pathomechanisms implicated in PCDH19-CE.     

Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review

Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve.     

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells

The hypoxia associated with the transforming growth factor-β2 (TGF-β2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O₂ gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-β2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-β2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-β2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-β2 increased proton leakage. The findings reported herein indicate that the TGF-β2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.     

Primary and Secondary Cone Cell Death Mechanisms in Inherited Retinal Diseases and Potential Treatment Options

Inherited retinal diseases (IRDs) are a leading cause of blindness. To date, 260 disease-causing genes have been identified, but there is currently a lack of available and effective treatment options. Cone photoreceptors are responsible for daylight vision but are highly susceptible to disease progression, the loss of cone-mediated vision having the highest impact on the quality of life of IRD patients. Cone degeneration can occur either directly via mutations in cone-specific genes (primary cone death), or indirectly via the primary degeneration of rods followed by subsequent degeneration of cones (secondary cone death). How cones degenerate as a result of pathological mutations remains unclear, hindering the development of effective therapies for IRDs. This review aims to highlight similarities and differences between primary and secondary cone cell death in inherited retinal diseases in order to better define cone death mechanisms and further identify potential treatment options.     

Protective Effect of Quercetin on Sodium Iodate-Induced Retinal Apoptosis through the Reactive Oxygen Species-Mediated Mitochondrion-Dependent Pathway

Age-related macular degeneration (AMD) leads to gradual central vision loss and is the third leading cause of irreversible blindness worldwide. The underlying mechanisms for this progressive neurodegenerative disease remain unclear and there is currently no preventive treatment for dry AMD. Sodium iodate (NaIO₃) has been reported to induce AMD-like retinal pathology in mice. We established a mouse model for AMD to evaluate the effects of quercetin on NaIO₃-induced retinal apoptosis, and to investigate the pertinent underlying mechanisms. Our in vitro results indicated that quercetin protected human retinal pigment epithelium (ARPE-19) cells from NaIO₃-induced apoptosis by inhibiting reactive oxygen species production and loss of mitochondrial membrane potential as detected by Annexin V-FITC/PI flow cytometry. We also evaluated the relative expression of proteins in the apoptosis pathway. Quercetin downregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP and upregulated the expression of Bcl-2 through reduced PI3K and pAKT expressions. Furthermore, our in vivo results indicated that quercetin improved retinal deformation and increased the thickness of both the outer nuclear layer and inner nuclear layer, whereas the expression of caspase-3 was inhibited. Taken together, these results demonstrate that quercetin could protect retinal pigment epithelium and the retina from NaIO₃-induced cell apoptosis via reactive oxygen species-mediated mitochondrial dysfunction, involving the PI3K/AKT signaling pathway. This suggests that quercetin has the potential to prevent and delay AMD and other retinal diseases involving NaIO₃-mediated apoptosis.     

Neuroprotection,Growth Factors and BDNF-TrkB Signalling in Retinal Degeneration

Neurotrophic factors play key roles in the development and survival of neurons. The potent neuroprotective effects of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF) and nerve growth factor (NGF), suggest that they are good therapeutic candidates for neurodegenerative diseases. Glaucoma is a neurodegenerative disease of the eye that causes irreversible blindness. It is characterized by damage to the optic nerve, usually due to high intraocular pressure (IOP), and progressive degeneration of retinal neurons called retinal ganglion cells (RGCs). Current therapy for glaucoma focuses on reduction of IOP, but neuroprotection may also be beneficial. BDNF is a powerful neuroprotective agent especially for RGCs. Exogenous application of BDNF to the retina and increased BDNF expression in retinal neurons using viral vector systems are both effective in protecting RGCs from damage. Furthermore, induction of BDNF expression by agents such as valproic acid has also been beneficial in promoting RGC survival. In this review, we discuss the therapeutic potential of neurotrophic factors in retinal diseases and focus on the differential roles of glial and neuronal TrkB in neuroprotection. We also discuss the role of neurotrophic factors in neuroregeneration.     

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells to Model Myogenesis and Muscle Regeneration

In vitro organoids derived from human pluripotent stem cells (hPSCs) have been developed as essential tools to study the underlying mechanisms of human development and diseases owing to their structural and physiological similarity to corresponding organs. Despite recent advances, there are a few methodologies for three-dimensional (3D) skeletal muscle differentiation, which focus on the terminal differentiation into myofibers and investigate the potential of modeling neuromuscular disorders and muscular dystrophies. However, these methodologies cannot recapitulate the developmental processes and lack regenerative capacity. In this study, we developed a new method to differentiate hPSCs into a 3D human skeletal muscle organoid (hSkMO). This organoid model could recapitulate the myogenesis process and possesses regenerative capacities of sustainable satellite cells (SCs), which are adult muscle stem/progenitor cells capable of self-renewal and myogenic differentiation. Our 3D model demonstrated myogenesis through the sequential occurrence of multiple myogenic cell types from SCs to myocytes. Notably, we detected quiescent, non-dividing SCs throughout the hSkMO differentiation in long-term culture. They were activated and differentiated to reconstitute muscle tissue upon damage. Thus, hSkMOs can recapitulate human skeletal muscle development and regeneration and may provide a new model for studying human skeletal muscles and related diseases.     

Visual Disfunction due to the Selective Effect of Glutamate Agonists on Retinal Cells

One of the causes of nervous system degeneration is an excess of glutamate released upon several diseases. Glutamate analogs, like N-methyl-DL-aspartate (NMDA) and kainic acid (KA), have been shown to induce experimental retinal neurotoxicity. Previous results have shown that NMDA/KA neurotoxicity induces significant changes in the full field electroretinogram response, a thinning on the inner retinal layers, and retinal ganglion cell death. However, not all types of retinal neurons experience the same degree of injury in response to the excitotoxic stimulus. The goal of the present work is to address the effect of intraocular injection of different doses of NMDA/KA on the structure and function of several types of retinal cells and their functionality. To globally analyze the effect of glutamate receptor activation in the retina after the intraocular injection of excitotoxic agents, a combination of histological, electrophysiological, and functional tools has been employed to assess the changes in the retinal structure and function. Retinal excitotoxicity caused by the intraocular injection of a mixture of NMDA/KA causes a harmful effect characterized by a great loss of bipolar, amacrine, and retinal ganglion cells, as well as the degeneration of the inner retina. This process leads to a loss of retinal cell functionality characterized by an impairment of light sensitivity and visual acuity, with a strong effect on the retinal OFF pathway. The structural and functional injury suffered by the retina suggests the importance of the glutamate receptors expressed by different types of retinal cells. The effect of glutamate agonists on the OFF pathway represents one of the main findings of the study, as the evaluation of the retinal lesions caused by excitotoxicity could be specifically explored using tests that evaluate the OFF pathway.     

Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.     

Early Functional Impairment in Experimental Glaucoma Is Accompanied by Disruption of the GABAergic System and Inceptive Neuroinflammation

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABA_A receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.     

Assessment of Normal Tissue Radiosensitivity by Evaluating DNA Damage and Repair Kinetics in Human Brain Organoids

DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids (“mini-brains”) from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.     

Molecular Mechanisms of Retinal Pigment Epithelium Dysfunction in Age-Related Macular Degeneration

The retinal pigment epithelium (RPE), situated upon Bruch’s membrane, plays multiple roles in the ocular system by interacting with photoreceptors and. Therefore, dysfunction of the RPE causes diseases related to vision loss, such as age-related macular degeneration (AMD). Despite AMD being a global cause of blindness, the pathogenesis remains unclear. Understanding the pathogenesis of AMD is the first step for its prevention and treatment. This review summarizes the common pathways of RPE dysfunction and their effect in AMD. Potential treatment strategies for AMD based on targeting the RPE have also been discussed.