Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells.
The neuropathology of Parkinson's Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.
Mathiasen
Joanne R
JR
Neurobiology, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA.
McKenna
Beth Ann W
BA
Saporito
Michael S
MS
Ghadge
Ghanashyam D
GD
Roos
Raymond P
RP
Holskin
Beverly P
BP
Wu
Zhi-Liang
ZL
Trusko
Stephen P
SP
Connors
Thomas C
TC
Maroney
Anna C
AC
Thomas
Beth Ann
BA
Thomas
Jeffrey C
JC
Bozyczko-Coyne
Donna
D
D015655:1-Methyl-4-phenylpyridinium / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D000818:Animals; D016466:CHO Cells; D002227:Carbazoles / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D015655:1-Methyl-4-phenylpyridinium; D002227:Carbazoles; D007211:Indoles; D020930:MAP Kinase Kinase Kinases; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C106592:3,9-bis((ethylthio)methyl)-K-252a