bims-mideyd 2023-10-22 papers

Neural Regen Res. 2024 Apr;19(4): 807-810

Cell replacement with stem cell-derived retinal ganglion cells from different protocols.

Glaucoma, characterized by a degenerative loss of retinal ganglion cells, is the second leading cause of blindness worldwide. There is currently no cure for vision loss in glaucoma because retinal ganglion cells do not regenerate and are not replaced after injury. Human stem cell-derived retinal ganglion cell transplant is a potential therapeutic strategy for retinal ganglion cell degenerative diseases. In this review, we first discuss a 2D protocol for retinal ganglion cell differentiation from human stem cell culture, including a rapid protocol that can generate retinal ganglion cells in less than two weeks and focus on their transplantation outcomes. Next, we discuss using 3D retinal organoids for retinal ganglion cell transplantation, comparing cell suspensions and clusters. This review provides insight into current knowledge on human stem cell-derived retinal ganglion cell differentiation and transplantation, with an impact on the field of regenerative medicine and especially retinal ganglion cell degenerative diseases such as glaucoma and other optic neuropathies.

Keywords: cell clumps; cell suspension; cell transplantation; differentiation; direct-induced protocol; glaucoma; optic neuropathy; regenerative medicine; retinal ganglion cell; retinal organoids; stem cells

DOI: https://doi.org/10.4103/1673-5374.381494

Mol Vis. 2023 ;29 87-101

The impact of early RPE cell junction loss on VEGF, Ang-2, and TIMP secretion in vitro.

Chase Paterson, Jamen Cannon, Elizabeth Vargis.

Purpose: The retinal pigment epithelium (RPE) is an important tissue for maintaining a healthy retina. Retinal pigment epithelial cells help regulate nutrient transport to photoreceptors and are heavily pigmented to prevent light scattering. These cells also have junction proteins to form monolayers. Monolayers are key players in pathologies such as age-related macular degeneration (AMD), a leading cause of vision loss in older adults. During AMD, RPE cell detachment can occur, resulting in a loss of junctions. Losing junctions can increase the expression of pro-angiogenic vascular endothelial growth factor (VEGF). This overexpression can cause abnormal blood vessel growth or angiogenesis in the retina. Age-related macular degeneration treatments target VEGF to slow angiogenesis progression. However, other proteins, such as angiopoietin-2 (Ang-2) and the tissue inhibitor of metalloproteinase-1 (TIMP-1), may also play important roles, making them potential targets for treatment. Controlling RPE junction formation will help elucidate the relationship between RPE cell detachment and additional angiogenic factor secretion, lead to more therapeutics, and increase the efficacy of current treatments.
Methods: Micropatterning was used to control the spatial arrangement of primary porcine RPE cells using polydimethylsiloxane (PDMS) stencils. Patterns were formed into PDMS stencils to mimic 10%, 25%, and 50% overall detachment of the RPE monolayer. Zonula-occludens-1 (ZO-1), Ang-2, and VEGF were visualized using immunocytochemical (ICC) staining. An enzyme-linked immunosorbent assay (ELISA) was used to quantify extracellular Ang-2, VEGF, TIMP-1, and TIMP-2 levels. A rod outer segment (OS) phagocytosis assay was performed to determine how RPE junction loss directly affects photoreceptor support.
Results: The growth of primary porcine RPE cells was successfully controlled using stencils. Morphological changes and a decrease in pigmentation were observed, showing a decline in barrier and light absorption functions as degeneration increased. One day after stencil removal, junction proteins were delocalized, and angiogenic factor secretions were correlated with increased levels of detachment. Secretion levels of Ang-2 and TIMP-1 were significantly increased, whereas VEGF and TIMP-2 concentrations were not as affected by varying levels of detachment. OS phagocytosis appeared lower in RPE cells when ZO-1 was affected.
Conclusions: These results suggest a correlation between loss of junctions, abnormal angiogenic protein secretion, and reduced OS phagocytosis. Furthermore, Ang-2 and TIMP-1 proteins might be beneficial targets for AMD treatments, and their roles in retinal diseases deserve further investigation.

Trends Neurosci. 2023 Oct 16. pii: S0166-2236(23)00224-2. [Epub ahead of print]

Lysosomes in retinal health and disease.

Patricia Boya, Kai Kaarniranta, James T Handa, Debasish Sinha.

Lysosomes play crucial roles in various cellular processes - including endocytosis, phagocytosis, and autophagy - which are vital for maintaining retinal health. Moreover, these organelles serve as environmental sensors and act as central hubs for multiple signaling pathways. Through communication with other cellular components, such as mitochondria, lysosomes orchestrate the cytoprotective response essential for preserving cellular homeostasis. This coordination is particularly critical in the retina, given its high metabolic rate and susceptibility to photo-oxidative stress. Consequently, impaired lysosomal function and dysregulated communication between lysosomes and other organelles contribute significantly to the pathobiology of major retinal degenerative diseases. This review explores the pivotal role of lysosomes in retinal cells and their involvement in retinal degenerative diseases.

Keywords: age-related macular degeneration; glaucoma; lysosome membrane permeabilization; lysosome–mitochondria crosstalk; mTOR signaling; retinitis pigmentosa

DOI: https://doi.org/10.1016/j.tins.2023.09.006

Mol Aspects Med. 2023 Oct 13. pii: S0098-2997(23)00057-2. [Epub ahead of print]94 101217

Molecular aspects of optic nerve autophagy in glaucoma.

Yasushi Kitaoka, Kana Sase.

The optic nerve consists of the glia, vessels, and axons including myelin and axoplasm. Since axonal degeneration precedes retinal ganglion cell death in glaucoma, the preceding axonal degeneration model may be helpful for understanding the molecular mechanisms of optic nerve degeneration. Optic nerve samples from these models can provide information on several aspects of autophagy. Autophagosomes, the most typical organelles expressing autophagy, are found much more frequently inside axons than around the glia. Thus, immunoblot findings from the optic nerve can reflect the autophagy state in axons. Autophagic flux impairment may occur in degenerating optic nerve axons, as in other central nervous system neurodegenerative diseases. Several molecular candidates are involved in autophagy enhancement, leading to axonal protection. This concept is an attractive approach to the prevention of further retinal ganglion cell death. In this review, we describe the factors affecting autophagy, including nicotinamide riboside, p38, ULK, AMPK, ROCK, and SIRT1, in the optic nerve and propose potential methods of axonal protection via enhancement of autophagy.

Keywords: AMPK; Autophagy; Glaucoma; Nicotinamide; Optic nerve; ROCK; ULK

DOI: https://doi.org/10.1016/j.mam.2023.101217

Proc Natl Acad Sci U S A. 2023 Oct 24. 120(43): e2301733120

The murine retinal pigment epithelium requires peroxisomal β-oxidation to maintain lysosomal function and prevent dedifferentiation.

Sai Kocherlakota, Yannick Das, Daniëlle Swinkels, Maarten Vanmunster, Manon Callens, Stefan Vinckier, Frédéric M Vaz, Debasish Sinha, Paul P Van Veldhoven, Marc Fransen, Myriam Baes.

Retinal pigment epithelium (RPE) cells have to phagocytose shed photoreceptor outer segments (POS) on a daily basis over the lifetime of an organism, but the mechanisms involved in the digestion and recycling of POS lipids are poorly understood. Although it was frequently assumed that peroxisomes may play an essential role, this was never investigated. Here, we show that global as well as RPE-selective loss of peroxisomal β-oxidation in multifunctional protein 2 (MFP2) knockout mice impairs the digestive function of lysosomes in the RPE at a very early age, followed by RPE degeneration. This was accompanied by prolonged mammalian target of rapamycin activation, lipid deregulation, and mitochondrial structural anomalies without, however, causing oxidative stress or energy shortage. The RPE degeneration caused secondary photoreceptor death. Notably, the deterioration of the RPE did not occur in an Mfp2/rd1 mutant mouse line, characterized by absent POS shedding. Our findings prove that peroxisomal β-oxidation in the RPE is essential for handling the polyunsaturated fatty acids present in ingested POS and shed light on retinopathy in patients with peroxisomal disorders. Our data also have implications for gene therapy development as they highlight the importance of targeting the RPE in addition to the photoreceptor cells.

Keywords: lipids; lysosomes; peroxisomes; retinal degeneration; retinal pigment epithelium

DOI: https://doi.org/10.1073/pnas.2301733120