Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Molecular, Cellular, and Developmental Biology

First Advisor

Anumantha G. Kanthasamy

Abstract

Manganese (Mn) is an essential trace element found in most living organisms. Chronic exposure to Mn has been linked to the pathogenesis of manganism, which displays neurological abnormalities somewhat similar to those associated with Parkinson's disease resulting from dysfunction of the extrapyramidal motor system within the basal ganglia. However, the exact cellular and molecular mechanisms underlying Mn induced neurotoxicity have not been defined. Oxidative stress mediated dopaminergic neuronal apoptosis is considered to be the prime mechanisms of Mn neurotoxicity. Thus, we sought to identify the genes that are altered during Mn exposure and that lead us to elucidate the mechanisms underlying Mn induced neurotoxicity. First, we used the Qiagen mouse apoptosis RT2 Profiler™ quantitative PCR array system to identify the genes susceptible to Mn exposure. We treated C57 black mice with 10 mg/kg Mn via oral gavage for 30 days. Afterwards, PCR apoptosis array was performed on substantia nigral tissues for 84 genes associated with apoptotic signaling. Interestingly, we found a significant downregulation of the tumor repressor gene p73 in Mn-treated substantia nigral tissues. Western blot analyses revealed that the p73 isoform protein lacking transactivation domain at N-terminus (ΔNp73) was downregulated from substantia nigral tissues of C57 black mice exposed to 30 mg/kg Mn for 30 days via gavage. To further characterize the functional role of Mn-induced p73 downregulation in Mn neurotoxicity, we examined the interrelationships between the effects of Mn on p73 gene expression and apoptotic cell death in an N27 dopaminergic neuronal model. Mn exposure to 300 μM downregulated dNp73 proteins in N27 dopaminergic neurons in a time-dependent manner, which consistently supports our animal study. We further determined that protein level of the Np73 was also reduced in primary striatal cultures in a dose-dependent manner. Furthermore, overexpression of Np73 conferred modest cellular protection against Mn-induced neurotoxicity. Secondly, we identified signal transducer and activator of transcription 5b (STAT5b) gene which was downregulated both in a time-dependent and dose-dependent manner during Mn exposure in N27 dopaminergic neuronal cells over 12 h span. However, STAT1 was relatively unaffected during Mn treatment, indicating isoform-specific effect of Mn on STAT5b. Consistent to N27 dopaminergic neuronal cell model, Mn exposure downregulated STAT5b expression in primary mouse striatal culture. Quantitative RT-PCR analyses showed Mn exposure induces downregulation of STAT5b expression at the transcriptional level as well. Moreover, Bcl-2, a well-known downstream target of STAT5b pathway, was also downregulated concomitantly during Mn exposure. Pretreatment of 20 uM Lactacystin failed to protect downregulation of STAT5b indicating STAT5b downregulation was independent of proteasomal degradation pathway. Pre-treatment of N-Acetyl Cystine (NAC) was shown to protect downregulation of STAT5b. In addition, treatment of MPP+ in N27 cells showed downregulation of STAT5b. These results support the hypothesis that Mn exposure mediates oxidative stress that induces downregulation of STAT5b. Overexpression of STAT5b cells protected N27 cells against Mn-induced neurotoxicity. Furthermore, overexpression of STAT5b protected mitochondria in N27 cells. Downregulation of STAT5b was recapitulated in substnatia nigra of C57 black mice model treated with Mn and MitoPark Parkinson's disease model. We also present that human lymphocytes show downregulation of STAT5b during Mn exposure, proposing a potential drug candidate for Mn-induced neurotoxicity and Parkinson's disease patients. Futhermore, we show that Mn exposure suppresses promoter activity of STAT5b in MN9D dopaminergic cells. To characterize the molecular mechanisms underlying STAT5b downregulation during Mn neurotoxicity, we examined the effects of 300 μM Mn exposure for the promoter analysis of STAT5b expression. We subcloned the STAT5b promoter 1 from mouse brain. Analysis of mouse STAT5b promoter from 2,000 nt upstream to 5,00 nt downstream region indicated that a proximal region near exon 1 contains the regulatory element in response to Mn exposure. Detailed mutational analyses of the putative transcription factor binding site revealed that a Sp1 like transcription factor binding sites near exon 1 may be required for the suppression of STAT5b in Mn-induced neurotoxicity. Two KLF binding sites exhibited to be transcription repressor that can respond to Mn exposure, whereas one Sp1 binding sites exhibited transcription activator which senses Mn exposure and reduces its activity. These data suggest Mn exposure alters the profiles of transcription factors to downregulate anti-apoptotic STAT5b signaling via an Sp1-like transcription factor-dependent mechanism in dopaminergic neurons, which may significantly contribute to Mn neurotoxicity. Taken together, our results suggest that Mn exposure compromises the expression of neuroprotective dNp73 and STAT5B in dopaminergic neurons for Mn-induced neurotoxicity, thereby exacerbating neuronal cell death (NIH grants ES10586, ES19267, NS74443).

DOI

https://doi.org/10.31274/etd-180810-4581

Copyright Owner

Dongsuk Kim

Language

en

File Format

application/pdf

File Size

178 pages

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