Approaches for detecting lysosomal alkalinization and impaired degradation in fresh and cultured RPE cells: Evidence for a role in retinal degenerations

Abstract

Lysosomes contribute to a multitude of cellular processes, and the pH of the lysosomal lumen plays a central mechanistic role in many of these functions. In addition to controlling the rate of enzymatic degradation for material delivered through autophagic or phagocytotic pathways, lysosomal pH regulates events such as lysosomal fusion with autophagosomes and the release of lysosomal calcium into the cytoplasm. Disruption of either the steady state lysosomal pH or of the regulated manipulations to lysosomal pH may be pathological. For example, chloroquine elevates the lysosomal pH of retinal pigmented epithelial (RPE) cells and triggers a retinopathy characterized by the accumulation of lipofuscin-like material in both humans and animals. Compensatory responses to restore lysosomal pH are observed; new data illustrate that chronic chloroquine treatment increases mRNA expression of the lysosomal/autophagy master transcription factor TFEB and of the vesicular proton pump vHATPase in the RPE/choroid of mice. An elevated lysosomal pH with upregulation of TFEB and vHATPase resembles the pathology in fibroblasts of patients with mutant presenilin 1 (PS1), suggesting a common link between age-related macular degeneration (AMD) and Alzheimer’s disease. While the absolute rise in pH is often small, elevations of only a few tenths of a pH unit can have a major impact on both lysosomal function and the accumulation of waste over decades. Accurate measurement of lysosomal pH can be complex, and imprecise measurements have clouded the field. Protocols to optimize pH measurement from fresh and cultured cells are discussed, and indirect measurements to confirm changes in lysosomal pH and degradative capacity are addressed. The ability of reacidifying treatments to restore degradative function confirms the central role of lysosomal pH in these functions and identifies potential approaches to treat diseases of accumulation like AMD and Alzheimer’s disease. In summary, various approaches to determine lysosomal pH in fresh and cultured cells, as well as the potential to restore pH levels to an optimal range, can help identify and repair pathologies associated with lysosomal defects in RPE cells and perhaps also suggest new approaches to treat lysosomal storage diseases throughout the body.

Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2

“…7,11 The molecular mechanisms underpinning CQ/HCQ retinopathy have been widely studied but are not fully understood. 41,42 Studies have reported that CQ and HCQ can cause distinctive abnormalities in the perifoveal photoreceptor inner segment/outer segment junction. 42 CQ and HCQ have also been proposed to induce RPE degeneration through several pathways including elevation of lysosomal pH, 41 increasing RPE permeability, 43 and binding to melanin.…”

“…41,42 Studies have reported that CQ and HCQ can cause distinctive abnormalities in the perifoveal photoreceptor inner segment/outer segment junction. 42 CQ and HCQ have also been proposed to induce RPE degeneration through several pathways including elevation of lysosomal pH, 41 increasing RPE permeability, 43 and binding to melanin. 44 Our recent study showed that OATP1A2 is expressed in human RPE cells and, more importantly, that it mediates the uptake of atROL, a critical step in the human visual retinoid cycle.…”

“…40,41 Signs may include a central dark area surrounded by a hypopigmented zone in the retina. 7,11 The molecular mechanisms underpinning CQ/HCQ retinopathy have been widely studied but are not fully understood.…”

Purines in the eye: Recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland

“…The elevation of cytoplasmic cAMP following stimulation of the adenosine A 2A receptor likely underlies this restoration of acidic lysosomal pH. Together, this implies that conversion of released ATP into adenosine by ectoenzymes may influence the production of lipofuscin and lipid oxidation in RPE cells (Guha et al, 2014). Whether the balance between ATP and adenosine influences the progression of diseases like age-related macular degeneration remains to be seen.…”

Regulation of Phagolysosomal Digestion by Caveolin-1 of the Retinal Pigment Epithelium Is Essential for Vision

“…Lysosomal pH Measurement-Lysosomal pH values in cells grown to confluence in 96-well plates were measured using LysoSensor Yellow/Blue DND-160 (Life Technologies) following a published protocol (31). Briefly, cells were loaded with 7.5 M LysoSensor Yellow/Blue DND-160 for 3 min at 32°C in DMEM, followed by washing with 20 mM HEPES buffer containing 145 mM KCl, 10 mM dextrose, and 1 mM MgCl 2 .…”