Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

Major

Toxicology

First Advisor

Anumantha Kanthasamy

Second Advisor

Arthi Kanthasamy

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. The etiology of PD is currently not fully understood, but strong evidence has existed pointing to gene-environmental interactions that contribute to various pathogenic mechanisms of neurodegeneration: oxidative stress, neuroinflammation, mitochondrial dysfunction, and epigenetic modulation. While cell signaling mechanisms underlying neurotoxic injury have been actively studied in recent years, the signaling molecules contributing to compensatory survival signaling are largely unknown. Recently, we reported that the secreted neuropeptide prokineticin-2 (PK2) is upregulated during early stages of neurotoxic stress induced by MPP+, and plays a major compensatory protective function in nigral dopaminergic neurons in vitro and in vivo. Thus, the goal of my thesis work was to determine the transcriptional regulation and translational relevance of PK2 in cell culture and animal models of PD.

In Chapter 2, we studied the transcriptional and molecular regulatory mechanisms of Parkinsonian toxicants MPP+ and manganese (Mn)-induced PK2 upregulation. In a set of experiments, we performed in silico analysis of the PK2 promoter and revealed the presence of early growth response-1 (EGR1), hypoxia inducible factor 1 (HIF1α), transcription factor E2F, and nuclear respiratory factor-1 (NRF1) putative binding sites. Importantly, we observed that MPP+ and Mn exposure increased HIF1α and EGR1 levels at early stages of neurotoxicity. Since overexpression of HIF1α or EGR1 upregulated PK2 expression, these studies suggest that Mn regulates PK2 expression via EGR1 or HIF1α-dependent pathway. A toxicologically relevant dose of Mn exposure by an oral route significantly upregulated global PK2 levels in the brain, especially in the substantia nigra with concomitant increases in HIF1α transcription factor. Taken together, the results in this chapter suggest that dopaminergic neurotoxicants upregulates PK2 levels to counter the early neurotoxic stress, and that Mn-induced upregulation of PK2 expression is transcriptionally regulated by EGR1, HIF1α and NRF1.

Building on the findings in chapter 2, the focus of Chapter 3 is the role of PK2 in Mn-induced Parkinsonism because the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. Here we found that the dopaminergic neurotoxicant Mn rapidly induce PK2 during the early stages of Mn neurotoxic stress in an N27 dopaminergic cell model. To better understand the functional role of PK2 upregulation, we created stable PK2-expressing dopaminergic cells by delivering PK2 myc-tagged cDNA into mouse dopaminergic MN9D cells. Interestingly, PK2-overexpressing cells exposed to Mn showed significant protection against neurotoxicity relative to vector control cells, suggesting a neuroprotective role for PK2 in dopaminergic neurons. The protective effect was both dose- and time-dependent. Furthermore, the PK2 receptor blocker PC7 attenuated the PK2-induced neuroprotective effects in PK2-overexpressing cells. Overexpressing PK2 protected against manganese-induced apoptosis as measured by Annexin 5 and caspase-3 activation. We also found that mitochondrial integrity was well maintained in PK2-overexpressing cells relative to vector cells following exposure to Mn. Additional results also showed that key proteins involved in mitochondrial functions, including BCL2, PGC1-α and TFAM levels, were preserved in PK2-overexpressing cells during neurotoxic stress. Collectively, our results suggest that neurotoxic insults upregulate PK2 in dopaminergic neurons to protect against the early stages of neurotoxicity. Finally, observed the effect of Mn treatment on PK2 expression using a GENSAT PK2 GFP transgenic mouse model. During early exposure to Mn, PK2 levels were significantly upregulated in the SN while slightly decreasing in striatum. Prolonged exposure to Mn (30 mg/kg for 30 days) significantly upregulated PK2 levels in the brain, especially in the substantia nigra. Interestingly, in the striatum, where Mn-induced cell death mainly occurs, decreased PK2 levels were noted. Significant amount of methylation of the PK2 promoter region is also observed in the striatum. Combined with cell culture studies, the differential regulation of PK2 in the striatum and substantia nigra might suggest a possible neuroprotective role of PK2 in the SN during early exposure to Mn. Taken together, the results in this chapter suggest that Mn upregulates PK2 levels to counter early neurotoxic stress in vitro and in vivo.

Despite a wealth of preclinical studies establishing neuroprotective and neurorestorative properties of glial cell-line derived neurotrophic factor (GDNF) in animal models of PD, a number of phase II clinical trials utilizing direct intracranial injection of GDNF protein and AAV-mediated Gdnf gene transfer did not achieve efficacy that was hoped for. Setbacks from recent clinical trials might prompt a rethinking of the strategy which focused on ectopic expression of GDNF targeted towards neurons. Devising strategies to elevate GDNF expression by means other than genetic manipulation is the current challenge. The pharmacologically modulated signaling pathways that are in crosstalk with GDNF or could induce its endogenous upregulation represent opportunities to fully harness the clinical benefits of GDNF, without the side effects associated with current methods for GDNF delivery. In Chapter 4, we show that GDNF has significant crosstalk with prokineticin signaling in astrocytes. A small molecule, IS20, could activate prokineticin signaling to induce secretion and expression of GDNF from in cultured astrocytes via activation of prokineticin receptor 1 (PKR1) preferentially expressed by astrocytes. Further, non-invasive administration of the blood-brain-barrier-permeable lipophilic IS20 through intranasal delivery could pharmacologically modulate GDNF levels in the nigrostriatal system of C57B/6 mice. Importantly, IS20 treatment yielded significant neuroprotective and neurorestorative effects in the MPTP and MitoPark mouse models of PD. Our results indicate that the full clinical benefits of GDNF could be leveraged by pharmacological modulation using IS20.

In summary, we showed that PK2 could protect against classic Parkinsonian toxicants MPP+ and Mn induced neurotoxicity in MN9D mouse dopaminergic neuronal cells. We characterized the transcriptional regulation of PK2 by analyzing the PK2 promoter, and the results revealed that transcriptional factors HIF1α, EGR1, and NRF1 contribute to basal and neurotoxicity-induced PK2 expression. While studying the PK2 neuroprotective mechanisms, we found a fundamental relationship between PK2 and GDNF signaling pathways in astrocytes. Activation of PK2 signaling upregulates GDNF levels in vitro and in vivo. The protective role and therapeutic potential of the PK2-PKR1-GDNF signaling axis was further confirmed using a PKR1 agonist, IS20, in a MPTP mouse model of neurodegeneration as well as in the MitoPark genetic mouse model of PD. Collectively, we show that PK2 can be transcriptionally induced by multiple pro-survival factors and that PK2 signaling activation is a protective compensatory response to neurodegeneration. Pharmacological modulation of PK2 could induce GDNF upregulation and offer neuroprotective effects in multiple mouse models of PD. Thus, PK2 signaling represents a therapeutic target with great potential for PD treatment.

Copyright Owner

Jie Luo

Language

en

File Format

application/pdf

File Size

248 pages

Included in

Toxicology Commons

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