Text

ETD_2858

http://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000056354

theses

Loss of dopaminergic neurons, oxidative stress, deficient bioenergetics and decreased levels of reduced glutathione (GSH) in the substantia nigra are biochemical hallmarks of idiopathic Parkinson’s disease (PD). The intent of this study was to test the hypothesis that disturbances in GSH and dopamine (DA) homeostasis are detrimental to dopaminergic neurons possibly leading to cell damage. Moreover GSH-DA interactions are essential for dopaminergic neuronal survival. Reduced levels of bioenergetics could lead to loss of DA sequestration leading to extra vesicular DA and DA metabolites which could affect cellular function. We observed uptake of reduced DA into intact mitochondria. Oxidation products of dopamine (DAQ) and its major metabolite 3, 4-dihydrophenylacetic acid (DOPAC-Q) inhibited mitochondrial electron transport chain (ETC) activities, specifically complexes I and III in both lysed and intact mitochondria. Substrate activated complex I was irreversibly inhibited while only GSH and not other antioxidants attenuated complex I inhibition, suggesting that inhibition was mediated via oxidized DA rather than ROS production. Reduced levels of GSH in PD brains are unexplained, however it is possible that oxidized products of excess extra vesicular DA and DOPAC could form adducts with GSH thus removing GSH from the cytosolic and mitochondrial pool. Glutaredoxin (Grx) is a GSH related enzyme that specifically uses GSH as reductant to deglutathiolate during recovery from an oxidative stress episode. The current study characterized regional cytoplasmic Grx (Grx-1) activity, protein and mRNA expression as well as regional mitochondrial Grx (Grx-2) mRNA from rat brain. The striatum had the lowest Grx-1 activity, protein and message, while Grx-2 message was higher in this region than in other brain regions. Two peaks of Grx-1 activity and protein were observed during development, one within the first post natal week and a second increase in older animals (7-18 months). Since many neurodegenerative and neuropsychiatric diseases are gender biased, Grx-1 activity was measured in female and male rats. No gender differences were observed between males and proestrous females. Grx-1 and Grx-2 mRNA was also measured in cultured mesencephalic rat brain neurons and astrocytes. Neurons expressed 4-fold greater Grx-1 and 14-fold higher Grx-2 than astrocytes. These findings show distinct age, region and cellular differences in Grx activity that may have relevance to neuropathological conditions. Overall, these studies demonstrated that a loss in DA homeostasis could lead to loss of ETC function and that GSH serves to protect mitochondria from loss of function due to DA exposure. A deficit in GSH, low Grx-1 activity and/or altered DA biodisturbution would likely render regions intrinsically high in oxidative stress, such as the SNpc and striatum, particularly susceptible to oxidative damage thus leading to disease pathology.

Ph.D.

Includes bibliographical references

Includes vita

by Alpa H. Gautam

doi:10.7282/T30C4VJX

ETD doctoral

The author owns the copyright to this work.

ETD

application/pdf

application/x-tar

2058240

dfa67ec8a45d616dc7c771ce44a7e94d0a8293bf