Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model

Abstract

Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

Keywords: high-content analysis; lysosomal pH; lysosomes; neurons; pH biosensor; phenotypic screening.

Figure 1

HTS flowchart for identifying lysosomal pH modulators.

Figure 2

Hit selection for lysosomal acidifiers. (A–C) Population-based analysis. (A) 2D plot of FIRE-pHLy ratio FC versus nuclei FC (i.e., cell count) for all DMSO negative controls (shown in red dots; n = 384 wells across all assay plates). (B) 2D FIRE-pHLy ratio FC versus nuclei FC plot for all test compounds (n = 1 per compound; 1835 total compounds). Green dots represent primary hit compounds and yellow dots represent toxic or inactive compounds. (C) Expanded inset of acidic hits from Figure 2B. Acidic hits were selected using nucleus FC ≤ 3SD and FIRE-pHLy ratio FC ≤ 3SD compared to controls. Compounds that artificially altered the FIRE-pHLy ratio FC through mCherry fluorescence were excluded (green dots with black cross marks). (D–H) Lysosomal object-based analysis. (D) 2D plot of median bin versus bin at max for DMSO negative controls (shown in red dots; n = 384 wells across all assay plates). (E) 2D plot of median bin versus bin at max for all the test compounds (shown in yellow dots; n = 1 per compound; 1835 total compounds). Green dots represent primary hit compounds. (F) Lysosomal object-based acidic hits from Figure 2E. Acidic hits were selected using median bin ≤ 3SD and bin at max ≤ 3SD. (Inset) Frequency distribution for the hit compound highlighted with a red circle. Gray bars represent negative control distribution. Red bars represent hit compound distribution. (G) Filtering hits for cell toxicity. 2D plot of median bin versus nucleus FC for all DMSO negative controls. (H) 2D plot of median bin versus nucleus FC for test compounds. Compounds highlighted in the red box were excluded due to cell toxicity; alkaline hits are highlighted by the blue box; and acidic hits are highlighted in the orange box. Compounds that altered mCherry fluorescence were excluded (green dots with black cross marks). (I) Venn diagram showing the overlap of final filtered alkaline and acidic hits selected from population-based and lysosomal object-based analyses. Data in this figure was visualized in DataWarrior.

Figure 3

Top acidic hits tested in differentiated and undifferentiated SH-SY5Y cells.

Figure 4

Hit selection summary. Summary of small molecule hits that modulate lysosomal pH in undifferentiated and differentiated SH-SY5Y cells.

Figure 5

OSI-027 and PP242 increases mature cathepsin D levels in SH-SY5Y cells and acidifies pH in human iAstrocytes. (A) Representative fluorescence images of undifferentiated SH-SY5Y cells treated with DMSO, 100 nM BafA1, 10 μM OSI-027, and 10 μM PP242 at t = 6 h and stained with BODIPY FL Pepstatin A probe. Scale bar = 10 μm. (B) Time course of cells treated with compounds for 0.5, 1, 1.5, 2, 4, and 6 h. Cells were incubated with BODIPY FL Pepstatin A for 30 min before live-imaging. BODIPY FL fluorescence was normalized to cell number, displayed as a FC relative to control, and plotted against time (hours). Data points are presented as mean ± SD, from 3 biological replicates; n = ∼15,000–20,000 cells quantified per condition group per time point. Statistical analysis was performed using two-way ANOVA for multiple comparisons. *p ≤ 0.05 and **p ≤ 0.01. (C) Bar graph quantification of FIREpHLy ratio fold-change (FC) in human iPSC-derived astrocytes (iAstrocytes) treated with OSI-027 and PP242 at 10 μM for 24 h. Data points are presented as median ± SD from three technical replicates. Statistical analysis was performed using one-way ANOVA for multiple comparisons. ***p ≤ 0.001.

Figure 6

OSI-027 and PP242 inhibits mTORC1/2 and activates autophagy markers. (A) Simplified schematic of the proposed mechanism for OSI-027 and PP242-mediated lysosomal acidification through autophagy (highlighted in red arrows). Compounds are shown in orange. (B) Representative immunoblots for mTORC1 and mTORC2 phosphorylation substrates P70S6K^Thr389 and Akt^Ser473, respectively, in FIRE-pHLy SH-SY5Y cells treated with OSI-027 and PP242 at 0.1, 1, and 10 μM. (C) Representative immunoblots for autophagy markers ULK1^Ser757, LC3B-I/LC3B-II, and p62 respectively, in FIRE-pHLy SH-SY5Y cells treated with OSI-027 (OSI) and PP242 (PP) (same as above). GAPDH was used as the housekeeping protein. Note: WT SH-SY5Y cells were used to generate the p62 immunoblots. (D) Bar graphs showing quantification of P70S6K^Thr389/total (E) Akt^Ser473/total, (F) ULK1^Ser757/total, (G) LC3B-II/LC3B-I, and (H) p62/GAPDH. OSI-027 shown in the top row and PP242 shown in the bottom row. Data is normalized to DMSO controls. Bars are presented as mean ± SD from three independent replicates. Statistical analysis was performed using one-way ANOVA for multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 7

Dose-response and time-course comparison of mTOR inhibitors on lysosomal acidification. Five-point dose response (10-fold serial dilution) from 0.0001 to 10 μM treatment of (A) OSI-027, (B) PP242, (C) Rapamycin, and (D) Torin1 measured after 2, 6, and 24 h in undifferentiated FIRE-pHLy expressing SH-SY5Y cells. FIRE-pHLy ratio measurements were normalized to dose- and time-matched controls. Data points are presented as mean ± SD, from 3 technical replicates; n = ∼15,000–20,000 cells quantified per condition group per time point.