Healthy Retinal Ganglion Cells Research Articles

Acquisition of neurodegenerative features in isogenic OPTN(E50K) human stem cell-derived retinal ganglion cells associated with autophagy disruption and mTORC1 signaling reduction

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research, with great potential for the use of hPSC-derived RGCs for studies of human retinal development, in vitro disease modeling, drug discovery, as well as their potential use for cell replacement therapeutics. Of all these possibilities, the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention, due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies, many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus, to further advance this field of research, in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein, which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK, along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs, and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together, these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1, contributing to the degeneration of RGCs.

Astrocytes modulate neurodegenerative phenotypes associated with glaucoma in OPTN(E50K) human stem cell-derived retinal ganglion cells.

SummaryAlthough the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.

Strategies on the application of stem cells based therapies for the treatment of optic neuropathies.

The retinal ganglion cells (RGCs) are not able to regenerate following optic nerve injury resulting in an irreversible vision loss in patients with optic neuropathies including glaucoma. Recent findings in ocular regeneration have opened promising avenues to apply stem cell-based modalities to restore vision in progressive optic neuropathies. Stem cell-based therapies can help to improve retinal regeneration by solving two major problems: (1) by preventing secondary degeneration of RGCs and preserving the remaining vision, and (2) by replacing degenerated RGCs and promoting RGC axon regeneration in the damaged area. The first approach, known as neuroprotective therapy, uses stem cells incorporated into the degenerating retina with an aim to offer a nourishing environment for damaged RGCs resulting in anatomic and functional improvement. The second approach, known as RGC replacement therapy, ultimately aims at replacing the damaged RGCs with healthy RGCs or RGC precursors (Gao et al., 2012; Fu et al., 2019) in order to restore the visual function. Both approaches are graphically represented in [Figure 1]. The implementation of cell replacement therapeutic approaches requires successful generation of clinically safe and functional RGCs in an environment where the transplants survive, appropriately integrate and engraft, as well as establish neurites within the hosts’ retina and direct consequent axons towards the relevant regions in the brain (Behtaj et al., 2020). In this work, we discuss the challenges that are required to be addressed prior to the implementation of stem cell-based therapies in clinical practice and, suggest potential solutions to overcome the current limitations.

Retinal Tissue Bioengineering, Materials and Methods for the Treatment of Glaucoma.

Glaucoma, a characteristic type of optic nerve degeneration in the posterior pole of the eye, is a common cause of irreversible vision loss and the second leading cause of blindness worldwide. As an optic neuropathy, glaucoma is identified by increasing degeneration of retinal ganglion cells (RGCs), with consequential vision loss. Current treatments only postpone the development of retinal degeneration, and there are as yet no treatments available for this disability. Recent studies have shown that replacing lost or damaged RGCs with healthy RGCs or RGC precursors, supported by appropriately designed bio-material scaffolds, could facilitate the development and enhancement of connections to ganglion cells and optic nerve axons. The consequence may be an improved retinal regeneration. This technique could also offer the possibility for retinal regeneration in treating other forms of optic nerve ailments through RGC replacement. In this brief review, we describe the innovations and recent developments in retinal regenerative medicine such as retinal organoids and gene therapy which are specific to glaucoma treatment and focus on the selection of appropriate bio-engineering principles, biomaterials and cell therapies that are presently employed in this growing research area. Identification of optimal sources of cells, improving cell survival, functional integration upon transplantation, and developing techniques to deliver cells into the retinal space without provoking immune responses are the main challenges in retinal cell replacement therapies. The restoration of visual function in glaucoma patients by the RGC replacement therapies requires appropriate protocols and biotechnology methods. Tissue-engineered scaffolds, the generation of retinal organoids, and gene therapy may help to overcome some of the challenges in the generation of clinically safe RGCs.

Impairment of Membrane Repolarization Accompanies Axon Transport Deficits in Glaucoma.

Glaucoma is a leading cause of blindness worldwide, resulting from degeneration of retinal ganglion cells (RGCs), which form the optic nerve. In glaucoma, axon transport deficits appear to precede structural degeneration of RGC axons. The period of time between the onset of axon transport deficits and the structural degeneration of RGC axons may represent a therapeutic window for the prevention of irreversible vision loss. However, it is unclear how deficits in axon transport relate to the electrophysiological capacity of RGCs to produce and maintain firing frequencies that encode visual stimuli. Here, we examined the electrophysiological signature of individual RGCs in glaucomatous retina with respect to axon transport facility. Utilizing the Microbead Occlusion Model of murine ocular hypertension, we performed electrophysiological recordings of RGCs with and without deficits in anterograde axon transport. We found that RGCs with deficits in axon transport have a reduced ability to maintain spiking frequency that arises from elongation of the repolarization phase of the action potential. This repolarization phenotype arises from reduced cation flux and K+ dyshomeostasis that accompanies pressure-induced decreases in Na/K-ATPase expression and activity. In vitro studies with purified RGCs indicate that elevated pressure induces early internalization of Na/K-ATPase that, when reversed, stabilizes cation flux and prevents K+ dyshomeostasis. Furthermore, pharmacological inhibition of the Na/K-ATPase is sufficient to replicate pressure-induced cation influx and repolarization phase phenotypes in healthy RGCs. These studies suggest that deficits in axon transport also likely reflect impaired electrophysiological function of RGCs. Our findings further identify a failure to maintain electrochemical gradients and cation dyshomeostasis as an early phenotype of glaucomatous pathology in RGCs that may have significant bearing on efforts to restore RGC health in diseased retina.

Crystallins in Retinal Ganglion Cell Survival and Regeneration

Crystallins are heterogeneous proteins classified into alpha, beta, and gamma families. Although crystallins were first identified as the major structural components of the ocular lens with a principal function to maintain lens transparency, further studies have demonstrated the expression of these proteins in a wide variety of tissues and cell types. Alpha crystallins (alpha A and alpha B) share significant homology with small heat shock proteins and have chaperone-like properties, including the ability to bind and prevent the precipitation of denatured proteins and to increase cellular resistance to stress-induced apoptosis. Stress-induced upregulation of crystallin expression is a commonly observed phenomenon and viewed as a cellular response mechanism against environmental and metabolic insults. However, several studies reported downregulation of crystallin gene expression in various models of glaucomatous nerodegeneration suggesting that that the decreased levels of crystallins may affect the survival properties of retinal ganglion cells (RGCs) and thus, be associated with their degeneration. This hypothesis was corroborated by increased survival of axotomized RGCs in retinas overexpressing alpha A or alpha B crystallins. In addition to RGC protective functions of alpha crystallins, beta and gamma crystallins were implicated in RGC axonal regeneration. These findings demonstrate the importance of crystallin genes in RGC survival and regeneration and further in-depth studies are necessary to better understand the mechanisms underlying the functions of these proteins in healthy RGCs as well as during glaucomatous neurodegeneration, which in turn could help in designing new therapeutic strategies to preserve or regenerate these cells.

Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage

To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N-methyl-D-aspartate (NMDA)-induced RGC damage. Bevacizumab, ranibizumab and pegaptanib were intravitreally injected each at two different concentrations. Respective vehicles of the three substances served as controls. In a different group, additionally a rat anti-VEGF antibody was injected after NMDA treatment. Retrogradely labelled RGC were counted on retinal wholemounts 1 week or 2 months after intravitreal introduction of the VEGF inhibitors. Electron microscopy (EM) was performed on normal rat eyes 2 months after introduction of the VEGF inhibitors. RGC counts in healthy rat eyes were essentially unchanged from those of the control animals after the administration of both low and high concentrations of bevacizumab, ranibizumab or pegaptanib. Compared to the other two substances, however, high doses of pegaptanib and its respective vehicle significantly decreased RGC after 1 week and led to a marked increase of mitochondrial swelling in EM. In eyes with NMDA-induced RGC damage, no changes of RGC numbers were detected after rat anti-VEGF antibody or bevacizumab, ranibizumab and pegaptanib at both tested concentrations. Even at higher doses, bevacizumab and ranibizumab showed no toxic effects on RGC in vivo in either untreated rats or in the NMDA-induced RGC damage model. Also a rat anti-VEGF antibody showed no adverse effects after NMDA. Anti-VEGF therapy therefore appears safe even for eyes with additional excitotoxic RGC damage. Potential harm from the pegaptanib carrier solution at very high local concentrations cannot be excluded.