Thapsigargin-induced autophagic flux impairment and inflammation are potentiated by CLN3 deficiency and alleviated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in human ARPE-19 cells.
Thapsigargin impairs autophagic flux and promotes inflammation in ARPE-19 cells, with these effects worsened by CLN3 deficiency and mitigated by AICAR.
Where it sits
this study against the rest of the aicar (acadesine) corpusSummary and findings
The study measured the effects of thapsigargin on autophagic markers and inflammatory cytokines in human ARPE-19 cells, particularly in the context of CLN3 deficiency. AICAR was tested for its ability to modulate these effects. The findings suggest that CLN3 deficiency exacerbates the impairment of autophagic flux and inflammation induced by thapsigargin.
Abstract
Juvenile neuronal lipofuscinosis (JNCL) is a rare disease caused by mutations in the CLN3 gene. It leads to early vision loss mediated by retinal degeneration. Impaired autophagosomal-lysosomal degradation is a major hallmark of JNCL pathology, and neuroinflammation has also been postulated to play a role in its pathogenesis. Thapsigargin, a selective inhibitor of sarco/endoplasmic reticulum Ca<sup>2+</sup>-ATPase, inhibits autophagy, leading to an accumulation of autophagosomes/autophagophores in cells. Cells with defective CLN3 protein function have been found to be particularly sensitive to the anti-autophagic effects of thapsigargin. Here, we characterized the effects of thapsigargin on inflammatory cytokines and autophagic markers in ARPE-19 cells using ELISA and western blotting. We further examined these effects in cells deficient in CLN3 function by exposing the cells to CLN3 siRNA and testing whether the effects of thapsigargin could be modulated by the well-known autophagy activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Thapsigargin induced the accumulation of LC3 and p62/SQSTM1, consistent with impaired autophagic flux in ARPE-19 cells. Additionally, we observed that thapsigargin possessed pro-inflammatory potential, as it induced the release of IL-6 in ARPE-19 cells, no inflammasome activation was detected. Both effects were enhanced by CLN3 siRNA and alleviated by AICAR. In conclusion, thapsigargin-induced impaired autophagic flux and the accompanying inflammatory response are more pronounced in CLN3-deficient ARPE-19 cells, indicating that loss of CLN3 function affects both autophagy and inflammatory signaling.
Background
This paper addresses the role of autophagy and inflammation in juvenile neuronal lipofuscinosis (JNCL), a condition linked to CLN3 gene mutations that leads to retinal degeneration. Prior research has established that impaired autophagy is a significant feature of JNCL pathology. Understanding how thapsigargin affects autophagy and inflammation in the context of CLN3 deficiency could provide insights into potential therapeutic strategies.
Methods
The study utilized human ARPE-19 cells to investigate the effects of thapsigargin on autophagic markers and inflammatory cytokines. The cells were treated with CLN3 siRNA to induce deficiency. The primary outcomes measured included the accumulation of autophagic markers (LC3 and p62/SQSTM1) and the release of IL-6, assessed through ELISA and western blotting.
Results
Thapsigargin induced the accumulation of LC3 and p62/SQSTM1, indicating impaired autophagic flux in ARPE-19 cells. Additionally, thapsigargin induced the release of IL-6, with both effects being enhanced by CLN3 siRNA and alleviated by AICAR. Specific numeric values for these findings were not reported in the abstract.
Interpretation
The findings suggest that CLN3 deficiency exacerbates the effects of thapsigargin on autophagy and inflammation, which aligns with previous literature on the role of autophagy in neurodegenerative diseases. However, the clinical significance of these effects remains uncertain, particularly given the in vitro nature of the study and the lack of human data. The limitations include potential confounds from the cell line used and the absence of longitudinal data.
Key findings
- Thapsigargin induced the accumulation of LC3 and p62/SQSTM1, indicating impaired autophagic flux.
- Thapsigargin induced the release of IL-6 in ARPE-19 cells.
- Both effects were enhanced by CLN3 siRNA and alleviated by AICAR.
Limitations
- In vitro study, results may not translate to human conditions.
- No numeric results reported in the abstract.
- Single cell line (ARPE-19) used, limiting generalizability.
- Short duration of exposure to treatments.