Date of Award
Joan C. Edwards School of Medicine
Type of Degree
Dr. Richard Egleton, Committee Chairperson
Dr. Kelley Kiningham
Dr. Richard Niles
Dr. Gary Rankin
Dr. Monica Valentovic
Methamphetamine (MA) neurotoxicity is particularly evident in the striatum where it causes extensive dopamine (DA) release and results in neurodegeneration. To identify specific signaling pathways and macromolecules involved in postsynaptic DA-induced striatal toxicity we used a SK-N-MC cell model that mimics postsynaptic D1 receptor-expressing striatal neurons. The cells were treated for 6-24 h with 0-50 µM DA. The concentration was chosen to impart physiological relevance to the study as it mirrors [DA] found within the striatum following MA exposure. We show that 25-50 µM DA resulted in protein changes consistent with nitro(oxidative) stress as well as enhanced cleavage of caspase (CASP) 9, CASP 3, and poly (ADP-ribose) polymerase (PARP). Despite elevated MnSOD protein there was not increased enzymatic activity. Examination revealed that MnSOD was nitrated following DA treatment. Exogenous pretreatment with polyethylene glycol superoxide dismutase, an antioxidant, prevented PARP cleavage. These results suggest a detrimental role for DA-induced reactive oxygen/nitrogen species (RO/NS). To differentiate the D1-mediated from D1-independent effects of DA-stimulated signaling and toxicity, the cells were treated with 0-50 µM SKF-38393, a D1 agonist; or pretreated with 10 µM SCH23390, a D1 antagonist. Both treatments resulted in significant PARP cleavage versus control, although D1 blockade was protective when compared to DA alone. Despite significant PARP fragmentation, treatment with the D1 agonist did not result in significant CASP3 cleavage suggesting D1 stimulation led to apoptosis in a manner independent of CASP3 activation. The MAP Kinase p38 was phosphorylated following D1 stimulation as well as D1-independent DA treatment. Inhibition of p38 with 2 µM SB203580 exacerbated CASP3 cleavage following D1 activation, but attenuated CASP3 and PARP fragmentation after D1 blockade. The contradictory results of p38 phosphorylation on CASP3 suggest that its mechanism of activation alters its downstream signaling from CASP3 stabilization to CASP3 fragmentation; thus, we have named p38 the “molecular switch” in this system. We assessed AP-1 reporter activity as it is a downstream target of p38 and a regulator of cell-life and –death. AP-1 transfected cells pretreated with a D1 antagonist had similar luciferase activity as DA, while stimulation of the D1 receptor did not enhance the reporter. Pretreatment with a p38 inhibitor reduced AP-1 activity, and AFos-, a cFos dominant negative, transfected cells eradicated PARP cleavage. The findings indicate that DA-induced AP-1 is a D1- independent, p38-mediated, cFos-dependent apoptotic pathway. We hypothesize that DA increases RO/NS and alters redox-sensitive signaling mechanisms which result in apoptosis; and by 1) blocking D1- together with p38-activation, 2) inactivating AP-1, or 3) providing antioxidants we can prevent DA-mediated apoptosis that is stereotypical of MA-induced neurodegeneration.
Neuropharmacology -- Research.
Nervous system -- Degeneration -- Research.
Dopamine -- Research.
Asbury, Melinda L., "Mechanisms of Dopamine-Induced Methamphetamine Neurotoxicity" (2020). Theses, Dissertations and Capstones. 1322.
Available for download on Thursday, December 15, 2022