Manipulation and Study of Gene Expression in Neurotoxin- Treated Neuronal PC12 and SH-SY5Y Cells for In Vitro Studies of Parkinson’s Disease

Abstract

Neuronal PC12 and SH-SY5Y cells are highly suitable in vitro models for study of the neurodegenerative mechanisms occurring in Parkinson's disease (PD). Differentiated PC12 and SH-SY5Y cells bear many similarities to the neuronal populations affected in PD, and they provide a convenient source of large amounts of homogeneous material for biochemical and molecular downstream applications. In the present review, we describe how to differentiate PC12 and SH-SY5Y cells into neuron-like cells and provide protocols for their transfection with plasmids and infection with viral particles to manipulate gene expression. We also describe how to treat neuronal PC12 and SH-SY5Y cells with the classical PD neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-pyridinium ion (MPP+). Finally, we give detailed methods for several downstream applications useful for the analysis of cell death pathways in PD.

The drug adaptaquin blocks ATF4/CHOP-dependent pro-death Trib3 induction and protects in cellular and mouse models of Parkinson's disease

“…Because ATF4 and CHOP are potential upstream regulators of Trib3 ( Ohoka et al, 2005 ; Ord and Ord, 2005 ; Han et al, 2012 ) and contribute to Trib3 regulation in PD models ( Aimé et al, 2015 ), we also examined ATF4 and CHOP/Ddit3 mRNA levels. Consistent with previous reports ( Ryu et al, 2002 ; Holtz and O’Malley, 2003 ; Ryu et al, 2005 ; Sun et al, 2013 ; Aimé et al, 2018 ), both 6-OHDA and MPP + significantly upregulated ATF4 ( Fig. 3B , E ) and CHOP/Ddit3 ( Fig.…”

“…4A – D ). Consistent with previous findings ( Aimé et al, 2015 ; Aimé et al, 2018 ), 6-OHDA alone elevated ATF4, CHOP and Trib3 proteins ( Fig. 4A – D ).…”

“…To evaluate the neuroprotective potential of AQ, we first assessed survival and cellular morphology in a well-characterized PD cellular model of PC12 cells differentiated into neuron-like cells with NGF ( Greene and Tischler, 1976 ) and treated with 6-OHDA ( Ryu et al, 2002 ; Sun et al, 2013 ; Aimé et al, 2015 ; Aimé et al, 2018 ). Cultures were treated with diluent (DMSO) or 0.1, 0.5, 1 and 5 μM AQ ± 150 μM 6-OHDA ( Fig.…”

“…PC12 cells were cultured as described previously ( Greene and Tischler, 1976 ; Aimé et al, 2015 ; Aimé et al, 2018 ) on plastic cell culture dishes coated with rat tail collagen (Roche). Non-differentiated PC12 cells were grown in RPMI 1640 cell culture medium supplemented with 10% heat inactivated horse serum (Sigma), 5% fetal bovine serum (FBS) and penicillin/streptomycin.…”

“…Trib3 is induced by a range of PD-relevant stresses ( Kiss-Toth, 2015 ) including robust transcriptional activation in 6-hydroxydopamine (6-OHDA) models of PD both in vitro ( Ryu et al, 2005 ; Aimé et al, 2015 ) and in vivo ( Kanaan et al, 2015 ). Such induction is observed before measurable cell death in cellular PD models, including neuronal PC12 cells and rat ventral midbrain dopaminergic (VM DA) neurons treated with 6-OHDA, 1-methyl-4-phenylpyridinium (MPP + ), or α-synuclein (αSYN) fibrils ( Ryu et al, 2005 ; Aimé et al, 2015 ; Aimé et al, 2018 ). Trib3 over-expression is sufficient to induce neuron apoptosis and Trib3 knockdown protects from 6-OHDA, MPP + and αSYN-induced death ( Aimé et al, 2015 ).…”