Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4^−/−Mice: Pharmacologic Approaches and Functional Recovery

Abstract

Elevation of lysosomal pH in RPE cells interferes with the degradation of outer segments and may contribute to the pathologies associated with A2E. Pharmacologic elevation of cAMP can restore an acid pH and improve degradative function.

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

“…Addition of A2E to cultured RPE cells increases the lysosomal pH, slows degradation of proteins, and inhibits the translocation of protons into the lysosomes and the degradation of outer segments (3,15). Lysosomal pH in the RPE cells of ABCA4 Ϫ/Ϫ mice is elevated compared with age-matched controls (20), consistent with the alkalinizing effect of A2E.…”

“…The human ARPE-19 cell line was obtained from American Type Culture Collection (Manassas, VA) and processed as described previously (20). In brief, cells were grown to confluence in 25-cm 2 primary culture flasks (Becton Dickinson) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium with 3 mM L-glutamine, 100 g/ml streptomycin, and 2.5 mg/ml Fungizone and/or 50 g/ml gentamicin and 10% fetal bovine serum (all Invitrogen, Carlsbad, CA).…”

“…A previous study found that stimulation of A 2A adenosine receptors or ␤-adrenergic receptors reacidified compromised RPE cell lysosomes; the coupling of both receptors to the G s protein implicated the second messenger cAMP (20). Cell-permeant chlorophenylthio-cyclic AMP (cpt-cAMP) reacidified lysosomes, and myristoylated protein kinase inhibitor prevented acidification, strongly implying a role for protein kinase A.…”

“…Administration of the lysosmotic compound chloroquine leads to the incomplete digestion of photoreceptor outer segments within RPE cells, an increase in lysosomal-associated organelles, and the accumulation of extracellular debris around adjacent Bruch's membrane and choriocapillaris (24,29). Similarly, tamoxifen raises lysosomal pH and slows outer segment clearance in RPE cells (20). In autosomal recessive Stargardt's disease, a mutation in the ABCA4 transporter leads to the accumulation of N-retinylidene-N-retinylethanolamine (A2E) in RPE cells (1,27).…”

Mutations in MFSD8, Encoding a Lysosomal Membrane Protein, Are Associated with Nonsyndromic Autosomal Recessive Macular Dystrophy

“…This showed that an elevated lysosomal pH interfered with degradation of outer segments and the contribution to the N-retinylidene-N-retinylethanolaminee associated pathologies. 57 Two general strategies recently were investigated for the treatment of individuals with NCL. The identification of a master regulator of lysosomal biogenesis and autophagy, transcription factor EB, has uncovered the adaptive behavior of lysosomes in response to environmental cues as starvation.…”