Proteasome inhibitor lactacystin induces cholinergic degeneration.
OBJECTIVE
Ubiquitin proteasome system dysfunction is believed to play an important role in the development of Parkinson's disease (PD), and almost all studies till now have mainly focused on the susceptibility of dopaminergic neurons to proteasome inhibition. However, in fact, there are many other types of neurons such as cholinergic ones involved in PD. In our present study, we attempt to figure out what effect the failure of ubiquitin proteasome function would execute on cholinergic cells in culture.


METHODS
We treated cholinergic cells in culture with various doses of lactacystin. Then MTT assay was used to evaluate the cellular viability and the AnnexinV-PI method was used to detect apoptosis. Both cellular soluble and insoluble polyubiquitinated proteins were detected by western blot. Furthermore, the mitochondrial membrane potential was analyzed using JC-1 and the intracellular production of reactive oxygen species (ROS) was determined using the fluorescent probe CM-H2DCFDA.


RESULTS
We found that low doses of lactacystin were enough to induce significant apoptotic cell death, disturb the mitochondrial membrane potential, and cause oxidative stress. We also found that the amounts of polyubiquitinated proteins dramatically increased with high doses, although the loss of cells did not increase accordingly.


CONCLUSIONS
Our results suggest that cholinergic cells are sensitive to ubiquitin proteasome system dysfunction, which exerts its toxic effect by causing mitochondrial dysfunction and subsequent oxidative stress, not through polyubiquitinated proteins accumulation.
Zhou
Hai-Yan
HY
Department of Neurology & Institute of Neurology, Ruijin Hospital affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China.
Tan
Yu-Yan
YY
Wang
Zhi-Quan
ZQ
Wang
Gang
G
Lu
Guo-Qiang
GQ
Chen
Sheng-Di
SD
D000109:Acetylcholine / Q000378:metabolism*; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D025822:Ubiquitin C / Q000378:metabolism
D017304:Annexin A5; D015853:Cysteine Proteinase Inhibitors; D017382:Reactive Oxygen Species; D025822:Ubiquitin C; C067713:lactacystin; D000109:Acetylcholine; D000111:Acetylcysteine