Effects of Wild‐Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l‐DOPA‐Induced Toxicity in Postnatal Midbrain Culture

Abstract

• Abstract: Mutations in the free radical-scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn-SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild-type (WT SOD) and mutant (Gly 93 -÷ Ala; G93A) Cu/Zn-SOD enzyme on the fate of postnatal midbrain neurons. Oneweek-old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than nontransgenic cultures. In contrast, 1-week-old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with L-DOPA. L-DOPA at 200 pM caused~-..50% loss of tyrosine hydroxylase (TH) -positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 pM L-DOPA did not cause any significant reductions in TH-positive or GABA neuron numbers in transgenic WT SOD cultures. L-DOPA at 50 pM had opposite effects, in that it significantly increased TH-positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against L-DOPA-induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn-SOD enzyme have inverse effects. As the spontaneous loss and L-DOPA-induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn-SOD enzyme kills cells by oxidative stress.

Programmed cell death in amyotrophic lateral sclerosis

“…Similar observations were subsequently made in transfected PC-12 cells (29) and in primary neurons grown from transgenic mice expressing mutant SOD1 (30). Collectively, these in vitro data have led many investigators to consider that mutant SOD1 may kill motor neurons by activating PCD, a term that we here use in the sense of cell death mediated by specific signaling pathways.…”

Dopamine-modified α-synuclein blocks chaperone-mediated autophagy

“…Both neuronal populations had native cytosolic dopamine levels beneath the detection limit of the technique (<100 nM). Exposure to l-DOPA at 100 μM, a level that produces cytosolic dopaminochrome and neuromelanin (33) and eventually kills dopaminergic neurons but not the GABAergic neurons (34), increased cytosolic dopamine in unlabeled, predominantly GABAergic neurons to 2.5 ± 0.6 μM (n = 20), while in dopaminergic neurons, free cytosolic dopamine reached 17.7 ± 2.7 μM (n = 34, P < 0.001); thus, the level of dopamine reached in the neuronal cytosol was nearly identical to the dopamine concentration used to produce the DA-α-syn in vitro (14 μM).…”

“…The cultures were incubated with 100 μM l-DOPA for 1 hour prior to and during the recordings. After background current subtraction, dopamine concentration at the pipette tip was calculated using calibration curves generated for carbon fiber electrodes with different exposed surfaces (34). The initial concentration of cytosolic dopamine was calculated using the neuronal cell body volume and the volume of the pipette tip estimated from photographs acquired before each recording.…”

“…Total cellular levels of catechol were quantified by high-performance liquid chromatography with an electrochemical detection method by comparing to known amount of standards as described before (20,34,36).…”

Bcl-2 expression by retrograde transport of adenoviral vectors with Cre-loxP recombination system in motor neurons of mutant SOD1 transgenic mice

“…Most studies on cell survival by Bcl-2 have reported using cell lines transfected with mutant SOD1 cDNA, 31,32 and a few using primary neuronal cultures. 33 In our experiment, we used primary neuronal cells with SOD1 mutation separated from wildtype neuronal cells. We wanted to compare the difference between primary neuronal cells with SOD1 mutation and those with the wild-type in protection by overexpression of Bcl-2 using various apoptotic stimuli and conditions.…”