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Published on: August 24, 2022
Multiple programmed cell death pathways are involved in N-methyl-N-nitrosourea-induced photoreceptor degeneration
Miriam Reisenhofer1, Jasmin Balmer, Rahel Zulliger
1Department of Ophthalmology, Inselspital, University of Bern, Freiburgstrasse 14, 3010, Bern, Switzerland.
Purpose:
To identify programmed cell death (PCD) pathways involved in N-methyl-N-nitrosourea (MNU)-induced photoreceptor (PR) degeneration.
Methods:
Adult C57BL/6 mice received a single MNU i.p. injection (60 mg/kg bodyweight), and were observed over a period of 7 days. Degeneration was visualized by H&E overview staining and electron microscopy. PR cell death was measured by quantifying TUNEL-positive cells in the outer nuclear layer (ONL). Activity measurements of key PCD enzymes (calpain, caspases) were used to identify the involved cell death pathways. Furthermore, the expression level of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), key players in endoplasmic reticulum (ER) stress-induced apoptosis, was analyzed using quantitative real-time PCR.
Results:
A decrease in ONL thickness and the appearance of apoptotic PR nuclei could be detected beginning 3 days post-injection (PI). This was accompanied by an increase of TUNEL-positive cells. Significant upregulation of activated caspases (3, 9, 12) was found at different time periods after MNU injection. Additionally, several other players of nonconventional PCD pathways were also upregulated. Consequently, calpain activity increased in the ONL, with a maximum on day 7 PI and an upregulation of CHOP and GRP78 expression beginning on day 1 PI was found.
Conclusions:
The data indicate that regular apoptosis is the major cause of MNU-induced PR cell death. However, alternative PCD pathways, including ER stress and calpain activation, are also involved. Knowledge about the mechanisms involved in this mouse model of PR degeneration could facilitate the design of putative combinatory therapeutic approaches.
Insights
N-methyl-N-nitrosourea (MNU) causes photoreceptor degeneration through apoptosis, endoplasmic reticulum stress, and calpain activation. Understanding these programmed cell death pathways in mice may guide future combination therapies for retinal diseases.
Area of Science:
- Ophthalmology
- Cell Biology
- Toxicology
Background:
- Photoreceptor (PR) degeneration is a hallmark of many retinal diseases.
- N-methyl-N-nitrosourea (MNU) is a chemical agent used to induce experimental retinal degeneration in animal models.
- Identifying the specific cell death pathways involved is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the programmed cell death (PCD) pathways implicated in MNU-induced photoreceptor degeneration in a mouse model.
- To investigate the roles of apoptosis, endoplasmic reticulum (ER) stress, and calpain activation in this process.
Main Methods:
- Adult C57BL/6 mice were injected with MNU (60 mg/kg) and observed for 7 days.
- Histological analysis (H&E, electron microscopy) and TUNEL assays quantified PR cell death.
- Enzyme activity assays (calpain, caspases) and qRT-PCR for CHOP and GRP78 identified PCD pathway involvement.
Main Results:
- MNU induced photoreceptor cell death, characterized by decreased outer nuclear layer (ONL) thickness and increased TUNEL-positive cells starting day 3 post-injection.
- Activated caspases (3, 9, 12) and calpain activity were significantly upregulated.
- Expression of ER stress markers (CHOP, GRP78) increased from day 1 post-injection.
Conclusions:
- Apoptosis is the primary mechanism of MNU-induced photoreceptor cell death.
- Alternative PCD pathways, including ER stress and calpain activation, also contribute significantly.
- This mouse model provides insights into complex cell death mechanisms relevant for designing combination therapies for retinal degeneration.
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