Study On The Role And Mechanism Of Regulated Endocrine - Specific Protein RESP18 In Parkinson 's Disease

RESP18(Regulated Endocrine Secretory Protein,18kDa) is identified as a dopamine-regulated intermediate pituitary transcript. It is a unique endoplasmic reticulum (ER) resident by distal degradation. RESP18is closely relevant to endocrine. It is distributed mainly in the endocrine tissues and the central nervous system. So far, researchers of RESP18focus on the expression patterns in rat and human, and its regulatory factors and pathways. Its functions in the brain especially in the nervous system disorders have rarely been reported.Parkinson’s disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons. ER stress (ERS), caused by accumulation of unfolded or misfolded proteins, is an important pathological mechanism of PD. In my thesis, the functions and mechanisms of RESP18and Resp18-c (a new isoform of RESP18cloned in our lab) in PD were studied. The thesis is divided into two parts.In the first part, the mouse dopaminergic MN9D cells were treated with MPP+,6-OHDA and Rotenone respectively as in vitro PD models. In MN9D cells treated with150μM MPP+for24h or150μM6-OHDA for9h, the transcriptions of RESP18and ERS protein CHOP were dramatically upregulated, while ER stress marker--BiP only increased in6-OHDA-treated cells. Rotenone didn’t alter the expression of RESP18. RESP18protein level in MN9D cells was significantly increased by MPP+/6-OHDA, but not Rotenone. The expressions of BiP and CHOP protein were enhanced only in6-OHDA-treated cells. In addition, XBP1and its spliced form-XBPls didn’t change in MPP+or6-OHDA-injuried cells. ERS inducers, thapsigargin (TG) and tunicamycin (TM) could increase mRNA and protein levels of BiP, but RESP18protein was decreased under TG or TM treatment. Salubrinal, an ERS inhibitor, significantly reduced MPP+/6-OHDA-induced cell death. The three neurotoxin drugs have distinct mechanisms in cultured dopaminergic cells.6-OHDA but not MPP+, significantly increases hall markers of ERS such as BiP; Rotenone is thought to inhibit mitochondrial complex I and increases reactive oxygen species (ROS) to induce cell apoptosis, while its impact in ERS has rarely been reported. We conclude that RESP18is involved in PD possibly by means different from typical ERS. In the second part, we tested the effect of altered RESP18expression on cell survival challenged with MPP+or6-OHDA. A6and R13are two Lenti-shRNA constructs with high RESP18silencing efficiency, while A6is more efficient in silencing. The outcome of MTT showed that A6-transfected cell clone showed increased BiP expression and alleviated MPP+-induced cell death, but did not resist6-OHDA injury. Furthermore, RESP18overexpression exacerbated MPP+/6-OHDA-induced cell death but had little impact on BiP expression. DAPI staining and TUNEL staining were used to evaluate the apoptosis of cells exposed to MPP+or6-OHDA. We found that6-OHDA but not MPP+-mediated cell death was apoptotic. RESP18/RESP18-c knockdown could not chang the apoptosis rate while RESP18overexpression increased6-OHDA-induced cell apoptosis. To sum up, downregulation of RESP18/RESP18-c can alleviate MPP+-induced cell death by increasing the expression of BiP; RESP18overexpression can aggravate MPP+/6-OHDA-induced cell death possibly by a meachanism uncorrelated with ERS.