Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

University of North Carolina at Chapel Hill

Mitochondrial dysfunction, leading to elevated levels of reactive oxygen species, is associated with the pathogenesis of neurodegenerative disorders. Rotenone, a mitochondrial stressor induces caspase-9 and caspase-3 activation leading proteolytic cleavage of substrate nuclear poly(ADP-ribose) polymerase (PARP). PARP cleavage is directly related to apoptotic cell death. In this study, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1, as a rotenone-induced Parkinsonia-onset biomarker. We report that the innate ketone body, Na-D-β-hydroxybutyrate (NaβHB) reduces markedly the incidence of synphilin-1 aggregation. Furthermore, our data reveal that the metabolic byproduct also prevents rotenone-induced caspase-activated apoptotic cell death in dopaminergic SH-SY5Y cells. Together, these results suggest that NaβHB is neuroprotective; it attenuates effects originating from mitochondrial insult and can serve as a scaffold for the design and development of sporadic neuropathies.

Nitrosative stress mediated misfolded protein aggregation mitigated by Na-D-β-hydroxybutyrate intervention.

Protein disulfide isomerase (PDI), an important endoplasmic reticulum-resident oxidoreductase chaperone can bind to estrogens as well as intact with its receptor proteins [i.e. estrogen receptors (ER) α and β]. It has been postulated that PDI also acts as an intracellular 17β-estradiol (E2)-binding protein that transports and accumulates E2 in live cells. Drop in E2 level promotes dissociation of E2 from PDI and released in cytosol; the released E2 can augment estrogen receptor-mediated transcriptional activity and mitogenic action in cultured cells by modulating the ERβ/ERα ratio. In this study, we observed rotenone-induced damage to PDI leads to significant increase in ERβ/ERα ratio by down-regulating ERα and up-regulating ERβ. We demonstrated that nitrosative stress induced disruption of the cellular estrogenic status can be prevented through diphenyl difluoroketone (EF24, curcumin analog) intervention by protecting PDI from reactive oxygen species (ROS)-induced damage. Together, our study suggests that both PDI and EF24 can play a vital role in maintaining cellular estrogenic homeostasis.

EF24 prevents rotenone-induced estrogenic status alteration in breast cancer.

Nitrosative stress mediated S-nitrosylation (SNO) of protein disulfide isomerase (PDI), a housekeeping oxidoreductase, has been implicated in the pathogenesis of sporadic Parkinson's (PD) and Alzheimer's (AD) 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 α-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 α-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 α-synuclein and α-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 antiapoptotic tributes, and directly interfered with SNO-PDI formation. Lastly, we demonstrate that EA can bind human serum albumin (HSA). These results collectively indicate that small molecules can provide a therapeutic foothold for overcoming Parkinson's through a prophylactic approach.

Ellagic acid mitigates SNO-PDI induced aggregation of Parkinsonian biomarkers.

Endoplasmic reticulum (ER) proteins including protein disulfide isomerase (PDI) are playing crucial roles in maintaining appropriate protein folding. Under nitrosative stress, an excess of nitric oxide (NO) radical species induced the S-nitrosylation of PDI cysteines which eliminate its isomerase and oxidoreductase capabilities. In addition, the S-nitrosylation-PDI complex is the cause of aggregation especially of the α-synuclein (α-syn) protein (accumulation of Lewy-body aggregates). We recently identified a potent antioxidant small molecule, Ferrostatin-1 (Fer-1), that was able to inhibit a non-apoptotic cell death named ferroptosis. Ferroptosis cell death involved the generation of oxidative stress particularly lipid peroxide. In this work, we reported the neuroprotective role of ferrostatin-1 under rotenone-induced oxidative stress in dopaminergic neuroblastoma cells (SH-SY5Y). We first synthesized the Fer-1 and confirmed that it is not toxic toward the SH-SY5Y cells at concentrations up to 12.5 μM. Second, we showed that Fer-1 compound quenched the commercially available stable radical, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), in non-cellular assay at 82 %. Third, Fer-1 inhibited the ROS/RNS generated under rotenone insult in SH-SY5Y cells. Fourth, we revealed the effective role of Fer-1 in ER stress mediated activation of apoptotic pathway. Finally, we reported that Fer-1 mitigated rotenone-induced α-syn aggregation.

The neuroprotective role of ferrostatin-1 under rotenone-induced oxidative stress in dopaminergic neuroblastoma cells.

Amyloid beta (Aβ) aggregation is generally associated with Alzheimer's onset. Here, we demonstrate that incubation of dopaminergic SH-SY5Y cells with an Aβ peptide fragment (an 11-mer composed of residues 25-35; Aβ (25-35)) results in elevated intracellular nitrosative stress and induces chemical mutation of protein disulfide isomerase (PDI), an endoplasmic reticulum-resident oxidoreductase chaperone. Furthermore, Aβ (25-35) provokes aggregation of both the minor and major biomarkers of Parkinson's disease, namely, synphilin-1 and α-synuclein, respectively. Importantly, fluorescence studies demonstrate that Aβ (25-35) triggers colocalization of these Parkinsonian biomarkers to form Lewy-body-like aggregates, a key and irreversible milestone in the neurometabolic cascade leading to Parkinson's disease. In addition, fluorescence assays also reveal direct, aggregation-seeding interactions between Aβ (25-35), PDI and α-synuclein, suggesting neuronal pathogenesis occurs via prion-type cross-transfectivity. These data indicate that the introduction of an Alzheimer's-associated biomarker in dopaminergic cells is proliferative, with the percolative effect exercised via dual, independent, Parkinson-pathogenic pathways, one stress-derived and the other prion-like. The results define a novel molecular roadmap for Parkinsonian transfectivity via an Alzheimeric burden and reveal the involvement of PDI in amyloid beta induced Parkinson's.

An 11-mer Amyloid Beta Peptide Fragment Provokes Chemical Mutations and Parkinsonian Biomarker Aggregation in Dopaminergic Cells: A Novel Road Map for "Transfected" Parkinson's.

Parijat Kabiraj

Department of Cell Biology and Physiology,
School of Medicine,
Department of Cell Biology and Physiology, School of Medicine

University of North Carolina at Chapel Hill

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Parijat Kabiraj

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Physiology,School of Medicine,Department of Cell Biology and Physiology, School of Medicine

University of North Carolina at Chapel Hill

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Department of Cell Biology and Physiology,
School of Medicine,
Department of Cell Biology and Physiology, School of Medicine