Date of Award

2014-01-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Mahesh Narayan

Abstract

Cerebral accumulation of amyloidogenic protein aggregates is most frequently observed in the pathogenesis of neurodegenerative diseases. Recent studies showed prion like spreading of beta amyloid (A beta) in Alzheimer's disease (AD) and alpha synuclein protein in Parkinson's disease (PD) brain. Failure or compromise to the chaperone activity of protein disulfide isomerase (PDI) is also been reported as a major factor of aggregate formation. Nitrosative stress mediated S-nitrosylation (SNO) of protein disulfide isomerase (PDI), a housekeeping oxidoreductase, has been implicated in the pathogenesis of sporadic PD and AD. Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species (ROS), is associated with the pathogenesis of neurodegenerative disorders and neuronal cell death. Rotenone and MPTP has traditionally been employed as mitochondrial stressor to induce ROS insult in cell line experiments. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1 (a Parkinsonin biomarker) as a function of rotenone insult. We report that the innate ketone body, Na-D-beta-hydroxybutyrate (NabHB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, both rotenone and MPTP induce caspase-9 and caspase-3 activation leading to proteolytic cleavage of substrate nuclear poly (ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. Our data reveal that NaβHB also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells.

Interaction of A beta (1-42) and alpha-synuclein has also been speculated in previous studies. However, the mechanism behind the alleged interaction is not clear. Beta amyloid (25-35) fragment can induce toxicity as of A beta (1-42) peptide fragment, and is capable of forming beta sheet stacked fibril. We hypothesized whether the 25-35 mer can induce alpha-synuclein and promotes the interaction between A beta (25-35) and alpha-synuclein in SH-SY5Y cell. We found that the addition of beta amyloid (25-35) promotes intracellular accumulation of Lewy body (LB)-like inclusions (synphilin-1: alpha-synuclein). We have also found the A beta (25-35) induces S-nitrosylation of PDI, and subsequent increase in A beta (25-35) and PDI co-localization in SH-SY5Y. Together, these results strongly suggest that A beta (25-35) oligomers aggravate the formation of LB-like inclusions through posttranslational modification of PDI, highlighting PDI as a potential therapeutic target of neurodegenerative diseases.

Previous cell line studies have indicated that SNO-PDI formation provokes synphilin-1 aggregation, the minor Parkinsonian biomarker protein. Yet no work exists investigating whether SNO-PDI induces alpha-synuclein aggregation, the major Lewy body constituent associated with Parkinson's pathogenesis. Here, we report that SNO-PDI formation is linked to the aggregation of alpha-synuclein and also provokes α-synuclein:synphilin-1 deposits (Lewy body-like debris) normally found in the PD brain. Furthermore, we have examined the ability of a small molecule, 2,3,7,8-Tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione (ellagic acid; EA) to scavenge NOx radicals and to protect cells from SNO-PDI formation via rotenone insult both, cell-based and cell-independent in vitro experiments. Furthermore, EA not only mitigates nitrosative-stress-induced aggregation of synphilin-1 but also alpha-synuclein and alpha-synuclein:synphilin-1 composites (Lewy-like neurites) in PC12 cells. Mechanistic analyses of the neuroprotective phenomena revealed that EA lowered rotenone-instigated reactive oxygen species (ROS) and reactive nitrogen species (RNS) in PC12 cells, imparted anti-apoptotic tributes and directly interfered with SNO-PDI formation. Lastly, we demonstrate that EA can bind human serum albumin (HSA). Together these results collectively indicate that small molecules can provide a therapeutic foothold for overcoming Parkinson's through a prophylactic approach.

Language

en

Provenance

Received from ProQuest

File Size

176 pages

File Format

application/pdf

Rights Holder

Parijat Kabiraj

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