Parkinson’s disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER stress proteins contribute to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies for PD. Both the accumulation of mutant -synuclein (Syn), which causes an inherited form of PD, and the inhibition of mitochondrial complex I function by PDinducing neurotoxin lead to ER stress. The critical involvement of ER stress in experimental models of PD supports its potential relevance to PD pathogenesis and led us to test the hypothesis whether the homocysteine-inducible ER protein (Herp), an ubiquitin-like domain (UBD) containing ER-resident protein, can counteract mutant Syn- and neurotoxin- induced pathological cascades. In the first part of my study I showed that knockdown of Herp aggravates ER stress-mediated cell death induced by PD-linked mutant Syn. Functionally, Herp plays iv a role in maintaining ER homeostasis by facilitating proteasome-mediated degradation of ER-resident Ca2+ release channels in a neuronal-like cell line expressing the mutant A53T-Syn. Deletion of UBD or pharmacological inhibition of the proteasomes abolishes the Herp-mediated stabilization of ER Ca2+ homeostasis. Furthermore, knockdown or pharmacological inhibition of ER Ca2+ release channels ameliorates ER stress suggesting that impaired homeostatic regulation of Ca2+ channels promotes a protracted ER stress with the consequent activation of ER stress-associated cell death pathways. Interestingly, sustained upregulation of ER stress markers and aberrant accumulation of ER Ca2+ release channels were detected in transgenic mutant A53T- Syn mice. These data establish a causative link between impaired ER Ca2+ homeostasis and chronic ER stress in the degenerative cascades induced by mutant A53T-Syn and suggest that Herp is essential for the resolution of ER stress through maintenance of ER Ca2+ homeostasis. Because oxidants and mitochondria-derived free radicals can target ER-based Ca2+ regulatory proteins and cause uncontrolled Ca2+ release that may contribute to protracted ER stress resulting in cell death, I next determined the impact of the PD causing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the precursor of 1-methyl-4-phenylpyridinium (MPP+ ) on ER functions. I demonstrated that knockdown of Herp renders dopaminergic cells vulnerable to MPP+ -induced toxicity by a mechanism involving upregulation of CCAAT/enhancer binding protein homologous protein (CHOP) and depletion of the ER Ca2+ store. Conversely, ectopic expression of Herp confers protection by blocking MPP+ -induced CHOP upregulation, ER Ca2+ store depletion and mitochondrial Ca2+ accumulation in a manner dependent on a functional v ubiquitin-proteasomal protein degradation pathway. Deletion of the UBD or treatment with a proteasomal inhibitor abolished the central function of Herp in ER Ca2+ homeostasis. Collectively, our findings suggest that approaches that aim to increase Herp levels or its ER Ca2+ -stabilizing action may prevent or ameliorate neuronal loss in PD. Though abnormal protein aggregates are characteristic features of the slowly progressive neurodegenerative disorders, they are also found in acute pathological states such as cerebral ischemia. The role of protein aggregation in neuronal pathology after brain ischemia is not clear. In the last part of my work, I show that transient focal ischemia induces the continuous accumulation of insoluble Syn and DJ-1, two proteins linked to early-onset PD, in vulnerable neurons from the onset of reperfusion until delayed neuronal death. Double immunocytochemical analysis reveals that Syn and DJ-1 are co-localized in inclusion-like structures in the vulnerable neurons of the lesioned cortices suggesting that DJ-1 is recruited into the Syn-containing inclusions and thereby precludes this neuroprotective protein from exercising its anti-oxidant and chaperone-like activities. Supporting this notion, knockdown of DJ-1 promotes Syn insolubility and renders neurons vulnerable to an ischemic insult whereas ectopic expression of DJ-1 ameliorates Syn -induced degenerative cascades and reverses ischemic neuronal injury. Furthermore, mice deficient in Syn exhibit significantly smaller infarcts and improved behavioral recovery after ischemia compared to nontransgenic mice. Ablation of Syn ameliorates the accumulation of insoluble DJ-1 and the ensuing oxidative damage following an ischemic insult. Taken together, our data show that aberrant accumulation of Syn plays a precipitating role in ischemic neuronal vi injury and suggest that PD-causing mutations in Syn and DJ-1 can worsen ischemic brain damage. In conclusion, these studies provide insights into the molecular cascade of Syninduced degeneration and may uncover novel therapeutic strategies for PD and stroke.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-2824 |
| Date | 01 January 2011 |
| Creators | Belal, Cherine |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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