Autophagy disruption reduces mTORC1 activation leading to retinal ganglion cell neurodegeneration associated with glaucoma.

Autophagy dysfunction has been associated with several neurodegenerative diseases including glaucoma, characterized by the degeneration of retinal ganglion cells (RGCs). However, the mechanisms by which autophagy dysfunction promotes RGC damage remain unclear. Here, we hypothesized that perturbation of the autophagy pathway results in increased autophagic demand, thereby downregulating signaling through mammalian target of rapamycin complex 1 (mTORC1), a negative regulator of autophagy, contributing to the degeneration of RGCs. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor adenosine monophosphate-activated protein kinase (AMPK), along with subsequent neurodegeneration in RGCs differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated variant of Optineurin (OPTN-E50K). Similarly, the microbead occlusion model of glaucoma resulting in ocular hypertension also exhibited autophagy disruption and mTORC1 downregulation. 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 highlight an important balance between autophagy and mTORC1 signaling essential for RGC homeostasis, while disruption to these pathways contributes to neurodegenerative features in glaucoma, providing a potential therapeutic target to prevent neurodegeneration.

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

Although 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.

Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics.

A major obstacle in the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning. We evaluated potential artifacts by exploring the effects of serum, comparing activity data and CAPA data, and assessing the impact of the chloroalkane tag and its linker chemistry. We also used viral transduction to expand CAPA to the nuclear compartment in epithelial and neuronal cell lines. Using this enhanced method, we measured a 48-h time course of nuclear penetration for a panel of chemically diverse modified RNAs. Moving forward, CAPA will be a useful tool for deconvoluting the complex processes of endosomal uptake, escape into the cytosol, and subcellular trafficking of oligonucleotide therapeutics in therapeutically relevant cell types.

Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids.

The development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.

Differential susceptibility of retinal ganglion cell subtypes in acute and chronic models of injury and disease.

Retinal ganglion cells (RGCs) are a heterogeneous population of neurons, comprised of numerous subtypes that work synchronously to transmit visual information to the brain. In blinding disorders such as glaucoma, RGCs are the main cell type to degenerate and lead to loss of vision. Previous studies have identified and characterized a variety of RGC subtypes in animal models, although only a handful of studies demonstrate the differential loss of these RGC subtypes in response to disease or injury. Thus, efforts of the current study utilized both chronic (bead occlusion) and acute (optic nerve crush, ONC) rat models to characterize disease response and differential loss of RGC subtypes. Bead occlusion and ONC retinas demonstrated significant RGC loss, glial reactivity and apoptosis compared to control retinas. Importantly, bead occlusion and ONC retinas resulted in differential subtype-specific loss of RGCs, with a high susceptibility for alpha- and direction selective-RGCs and preferential survival of ipRGCs. Results of this study serve as an important foundation for future experiments focused on the mechanisms resulting in the loss of RGCs in optic neuropathies, as well as the development of targeted therapeutics for RGC subtype-specific neuroprotection.

Differentiation of retinal organoids from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) possess the remarkable ability to differentiate into any cell type of the body, including those of the retina. Through the differentiation of these cells as retinal organoids, it is now possible to model the spatial and temporal development of the human retina using hPSCs, in which retinal progenitor cells produce the entire repertoire of retinal cells, first differentiating into retinal ganglion cells and ending with mature photoreceptors, bipolar cells, and Müller glia. Importantly, retinal organoids self-assemble into laminated structures that recapitulate the layering of the human retina with a retinal ganglion cell layer lining the inner layer and a distinctly separate photoreceptor layer occupying the outer layers. This organoid technology has provided access to human tissue for developmental and disease modeling, as well as translational applications such as high throughput drug screening and cell replacement therapies. However, the differentiation of retinal organoids does require some expertise and multiple strategies produce inconsistent results. Here, we describe in detail a well-established and relatively simple method for the generation of retinal organoids.

Retinal Ganglion Cells With a Glaucoma OPTN(E50K) Mutation Exhibit Neurodegenerative Phenotypes when Derived from Three-Dimensional Retinal Organoids.

Retinal ganglion cells (RGCs) serve as the connection between the eye and the brain, with this connection disrupted in glaucoma. Numerous cellular mechanisms have been associated with glaucomatous neurodegeneration, and useful cellular models of glaucoma allow for the precise analysis of degenerative phenotypes. Human pluripotent stem cells (hPSCs) serve as powerful tools for studying human disease, particularly cellular mechanisms underlying neurodegeneration. Thus, efforts focused upon hPSCs with an E50K mutation in the Optineurin (OPTN) gene, a leading cause of inherited forms of glaucoma. CRISPR/Cas9 gene editing introduced the OPTN(E50K) mutation into existing lines of hPSCs, as well as generating isogenic controls from patient-derived lines. RGCs differentiated from OPTN(E50K) hPSCs exhibited numerous neurodegenerative deficits, including neurite retraction, autophagy dysfunction, apoptosis, and increased excitability. These results demonstrate the utility of OPTN(E50K) RGCs as an in vitro model of neurodegeneration, with the opportunity to develop novel therapeutic approaches for glaucoma.

Glaucoma as a Neurodegenerative Disease Caused by Intrinsic Vulnerability Factors.

Glaucoma, one of the most common causes of blindness in developing countries today, involves a progressive loss of neural cells in the optic nerve that leads to progressive, irreversible vision loss. Increased intraocular pressure (IOP) presents as a major risk factor for glaucoma, although there exist cases of glaucoma patients with normal IOP that exhibit damage to retinal ganglion cells (RGCs) and the optic nerve. However, treatment approaches have maintained their focus on modifying IOP due to a lack of other modifiable risks factors. Traditional concepts in glaucoma involve the neuronal environment and external effects as a source of causative factors; however, studies have yet to investigate whether the molecular profile of RGCs in glaucoma patients makes them more vulnerable and/or susceptible to external damage. Our hypothesis states that molecular changes at the whole cell, gene expression, and electrophysiological level of the neurons can contribute to their degeneration. Herein, we briefly describe different types of glaucoma and any similarities to different molecular and cellular features of neurodegeneration. To test our hypothesis, we describe human induced pluripotent stem cells (hiPSCs) as a reliable cellular tool to model neurodegenerative aspects of glaucoma to reveal the multiple pathological molecular mechanisms underlying disease development.

Astrocytes Regulate the Development and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells.

Retinal ganglion cells (RGCs) form the connection between the eye and the brain, with this connectivity disrupted in numerous blinding disorders. Previous studies have demonstrated the ability to derive RGCs from human pluripotent stem cells (hPSCs); however, these cells exhibited some characteristics that indicated a limited state of maturation. Among the many factors known to influence RGC development in the retina, astrocytes are known to play a significant role in their functional maturation. Thus, efforts of the current study examined the functional maturation of hPSC-derived RGCs, including the ability of astrocytes to modulate this developmental timeline. Morphological and functional properties of RGCs were found to increase over time, with astrocytes significantly accelerating the functional maturation of hPSC-derived RGCs. The results of this study clearly demonstrate the functional and morphological maturation of RGCs in vitro, including the effects of astrocytes on the maturation of hPSC-derived RGCs.