An 11-mer Amyloid Beta Peptide Fragment Provokes Chemical

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A 11-mer amyloid beta peptide fragment provokes chemical mutations and Parkinsonian biomarker aggregation in dopaminergic cells: a novel roadmap for “transfected” Parkinson’s Parijat Kabiraj, Jose Eduardo Marin, Armando Varela-Ramirez, and Mahesh Narayan ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00159 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 24, 2016

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ACS Chemical Neuroscience

A 11-mer amyloid beta peptide fragment provokes chemical mutations and Parkinsonian biomarker aggregation in dopaminergic cells: a novel roadmap for “transfected” Parkinson’s Parijat Kabiraj1, Jose Eduardo Marin2, Armando Varela-Ramirez2, Mahesh Narayan1* 1

Department of Chemistry, 2Department of Biological Sciences, Bioscience Research Building,

Border Biomedical Research Center, the Cytometry, Screening and Imaging Core Facility, University of Texas at El Paso, El Paso, Texas 79968 *Correspondence: [email protected]

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ABSTRACT Amyloid beta (Aβ) aggregation is generally associated with Alzheimer’s onset. Here, we demonstrate that incubation of a dopaminergic SH-SY5Y cells with an Aβ peptide fragment (an 11-mer comprised 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 viz., 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.


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KEYWORDS Neurodegeneration, Amyloid beta (25-35) peptide, alpha synuclein, synphilin-1, Lewy body, Protein disulfide isomerase.



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INTRODUCTION The progress of protein-misfolding-related neurodegenerative disorders is synonymous with the accumulation of disease-specific biomarkers. Amyloid beta (Aβ) deposits are associated with Alzheimerization of the brain and eventually to Alzheimer’s disease (AD), prion aggregates in Creutzfeldt-Jakob disease, Huntingtin load in Huntington’s disease and α-synuclein and synphilin-1 debris in Parkinson’s disease (PD) are established examples of proteins associated with their respective disorders.1-3 Molecular level investigations have recently revealed upstream chemospecific signatures along the trajectory leading to biomarker accumulation and neurodegenerative-disease onset. For example, elevated levels of reactive nitrogen species (RNS) and the resulting S-nitrosylation of protein cysteines have emerged as common causals/metabolic denominators in sporadic variants of several neurodegenerative disorders.4-6 In particular, chemical mutation of the oxidoreductase chaperone protein disulfide isomerases’ (PDI) active site cysteines by the potent gasotransmitter NO to form SNO-PDI has been convincingly implicated in Parkinson’s. 7-12 PDI, an endoplasmic reticulum (ER)-resident oxidoreductase chaperone that catalyzes post-translational maturation of disulfide bonds and facilitates ER trafficking has emerged as a critical intracellular neuroprotectant. 7,8,13. SNO-PDI formation has become increasingly recognized as a Rosetta-stone event in several other neuropathies including Amyotrophic Lateral Sclerosis (ALS) and AD.7-20 SNitrosylation of proteins other than PDI is also an important milestone in the pathophysiological cascades impacting synapse function, mitochondrial role, iron homeostasis and cell signaling.21 Other research has underscored macroscopic-level data supporting the role of PDI and chemical stress in AD pathophysiology. Examples include the co-localization of PDI with neurofibrillary tangles and cell-line studies wherein human PDI has been found to interact with endogenous human Tau on the ER.22,23,24 While elevated levels of RNS were found to contribute to altered Aβ metabolism the neuroprotective role of native (non-S-nitrosylated) PDI was clearly highlighted by its ability to prevent Aβ aggregates.25 In ALS pathology, PDI has been implicated via discovery of its expression in conjunction with the ER stress-marker ERp57 in mouse spines and cerebrospinal fluid. 25,26 Additionally, PDI in ALS is found to be S-nitrosylated, affecting its neuroprotective role and sharing common mechanistic hallmarks with AD.4 4 ACS Paragon Plus Environment

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SNO-PDI formation due to nitrosative stress also contributes to PD pathogenesis. Cellline work has revealed that SNO-PDI formation leads to the accumulation of polyubiquitinated proteins and activates the unfolded protein response.4,8,17 The NO-driven chemi-mutation of PDI cysteines triggers aggregation of PD-specific biomarkers, whereas overexpression of non-Snitrosylated PDI diminishes the occurrence of synphilin-1 inclusions and restores its freelydiffused cytosol distribution.14,15 These data reaffirm the neuroprotective role of PDI against RNS-induced PD pathogenesis. Related studies revealed that the presence of small-molecules that can scavenge RNS reduce SNO-PDI formation and inhibit the aggregation of PD biomarkers; laying a prophylactic foundation-stone against RNS-induced pathogenesis.11,18 Reports have suggested that that there is a risk of inter-neurodegenerative disease propagation by both amyloidogenic and Parkinsonian biopolymers due to their prion-like behavior/cross-reactivity.27,28 For example, Aβ in cerebrospinal fluid was determined to be a prognosticative biomarker for Parkinson’s.27,28 Yet, a gap remains in delineating with molecularlevel-precision how one neurodegenerative disease-linked biomarker may cross-fertilize another neurodegenerative process, heretofore considered unrelated to the initiating biomarker. An atomic/molecular-level resolution of the mechanism(s) by which disease-associated pathways can cross disease-specific boundaries is of fundamental and therapeutic interest. While the predominant forms of amyloid beta remain Aβ (1-40) and Aβ (1-42), a peptide fragment, viz. Aβ (25-35) has been shown to reproduce the cardinal features of Aβ in several experimental models leading to AD.29 Here, we have examined the effect of Aβ (25-35) insult on a dopaminergic neuroblastoma cell-line model. Our results reveal that upon Aβ (25-35) insult, RNS levels became elevated and PDI becomes increasingly S-nitrosylated. Aβ peptide incubation also triggers the apoptotic pathway in the cell-line and provokes the aggregation of Parkinson’s-specific biomarkers synphilin-1 and α-synuclein by two unrelated, and heretofore unknown, mechanisms.29,30 Importantly, the end-point of Aβ insult is the co-localization of synphilin-1 with α-synuclein to form Lewy-like bodies; an irreversible and milestone event in PD pathogenesis.

The results define the chemical footprint by which a particular

neurodegeneration-specific protein initiates a neuropathobiological outcome not directly associated with it.



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RESULTS AND DISCUSSION RNS mediated apoptosis upon Aβ (25-35) insult. Aβ (25-35) readily aggregated when introduced into distilled water as previously demonstrated (Figure 1A).31 The average diameter of the aggregates was 162 nm ± 37.32. The molecular weight of the aggregates was estimated to be 9.79 kDa compared to the monomeric molecular weight of 1060.3 Da as reported.31 The SH-SY5Y cell line used in this study remains an established model for mimicking dopaminergic neuronal cells and Parkinson’s progress.11 We examined the cytotoxicity of Aβ (25-35) by flow cytometry (Figure 1 B, C). The percentage cell-death increased in a dosedependent manner from ~11% at 1 µM to ~42% at 50 µM Aβ (25-35) (after 48 h incubation at 37ºC). A calculated cytotoxic concentration 50% (CC50) for Aβ (25-35) in SH-SY5Y cells was found to be 62 µM (CC50 is defined as the concentration of Aβ (25-35) needed to disrupt the integrity of the cellular membrane of 50% of the cell population after 48 h of treatment). Quantitative analysis of these data suggests that aggregated Aβ (25-35) is toxic even at concentrations as low as 5 µM (Figure 1C; P=0.0069). Expectedly, higher concentrations such as 50 µM resulted in a significantly increased cell-death relative to controls (P=0.0003).31 The findings indicate that Aβ (25-35) induces cellular toxicity in a dose-dependent manner. The mechanism (necrosis vs apoptosis) by which Aβ (25-35) insult results in cell-death was also examined (Figure 1, D-G). Cells treated with distilled H2O (0.1% v/v solvent control), 1 µM Aβ (25-35), or left untreated did not show any substantial increase in either necrosis or apoptosis [Fig 1 D, E]. However, the difference in results between 1 µM and 50 µM Aβ (25-25) treatment conditions was found to be statistically significant (P=0.0309) in the annexin V-FITC positive cells, due to phosphatidylserine externalization which is representative of apoptotic cell population (Figure 1E). Similar to cytotoxicity data, 5 µM Aβ (25-35) administration induced a significant increase of apoptotic pathway activity after 24 h relative to controls (P=0.026). The necrotic cell count after 24 h treatment is not high in any condition and is visible only after 48 h of exposure to Aβ (25-35) explaining the lack of necrotic cells in Figure 1E. Representative graphs of cytotoxicity and apoptosis/necrosis are shown in Figure 1B and 1D, respectively. The cleavage of Poly(ADP-ribose) polymerase-1 (PARP-1), a DNA repair enzyme responsible for the maintenance of genome integrity, is a hallmark of apoptosis.18 Native PARP1 cleavage was assessed via western blotting (Figure 1G). SH-SY5Y cells were treated with 1 6 ACS Paragon Plus Environment

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µM and 50 µM of Aβ (25-35) for 24 h and densitometric analysis of Western blots exhibited ~25% increment of PARP-1 cleavage compared to untreated controls (P=0.0092). PARP-1 cleavage data further support flow cytometry results (Figure 1D) monitoring phosphatidylserine externalization, indicating that Aβ (25-35) induces cytotoxicity via apoptosis.5,27 In addition, we examined any Aβ (25-35) dose dependent modulation of RNS and ROS levels in the SH-SY5Y cell-line via DAF-FM and DCF-DA assays, respectively (upon 24 h exposure to different doses of the peptide) (Figure 1H-J).31 The DAF-FM assay revealed a peptide-dose-dependent increase in RNS levels (Figure 1H and I). Confocal microscopy image analysis confirmed increased intracellular RNS production (Figure 1H) as a function of Aβ (2535) treatment levels (panel I, untreated; panel ii, 1µM; panel iii, 10 µM; panel iv, 20 µM). The increase in the intracellular RNS level is almost significant when untreated vs 20 µM Aβ (25-35) treatments are compared (P=.0573). By contrast, there was no detectable increase in ROS production even upon treatment with 50 µM Aβ (25-35) for 24 h (Figure 1J; P=0.3612). These data suggest that the increase in apoptotic activity in SH-SY5Y cells upon Aβ (25-35) treatment is associated with increased levels of RNS.

Role of Aβ (25-35) in synphilin-1 and α-synuclein aggregation. The major Parkinsonian biomarker α-synuclein is associated with the synthesis of dopamine.32 It’s co-localization with synphilin-1, a minor Parkinsonian biomarker, to form Lewy neurites represents a cardinal milestone in Parkinson’s pathogenesis.18,33 Interestingly, both Aβ (25-35) and α-synuclein, are categorized as amyloidogenic proteins where a dominantly alpha-helical functional form is transformed into a predominantly beta-sheet-containing rogue.3,28,34 Therefore, it is of interest to define the native-state distributions of α-synuclein and synphilin-1 in the presence of Aβ peptide. SH-SY5Y cells were transiently transfected or co-transfected (1:1) with synphilin-1 and/or α-synuclein. After Aβ (25-35) treatment, the SH-SY5Y cell line showed increased cytosolic protein aggregation (white arrow) of both, synphilin-1 (Figure 2A) and α-synuclein (Figure 2C) proteins. In case of (EGFP-tagged) synphilin-1, no significant change in intensity was observed up to 1 µM Aβ (25-35) treatment relative to the untreated condition (P=0.1456). However, upon incubation with 10 µM and 20 µM Aβ (25-35), synphilin-1 intensity significantly increased relative to control (P=0.0075; Figure 2B). There is also a significant



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difference in intensity when comparing 1 µM and 10 µM Aβ (25-35) treatment for 24 h (P=0.0466; Figure 2B). The effect of Aβ (25-35) on α-synuclein expression was more pronounced. Cell treatment with 1 µM Aβ (25-35) for 24 h significantly increased the intensity of α-synuclein protein (Figure 2D; P=0.0003). The maximum intensity of EGFP-synphilin-1 and α-synuclein proteins upon 20 µM Aβ (25-35) treatment were 80 a.u. and 120 a.u., respectively. This difference, ~ 50%, between the intensity of these two Parkinsonian proteins is conserved across essentially identical treatment conditions. These data suggest that Aβ (25-35) preferentially cross-reacts with α-synuclein over (EGFP-) synphilin-1. Next, we investigated whether Aβ (25-35) insult provokes the co-localization of the major and minor Parkinsonian biomarkers to form corresponding Lewy-like neurites. SH-SY5Y cells were co-transfected (1:1; DNA concentration) with synphilin-1 and α-synuclein using pEGFP-synphilin-1 plasmid and pCMV6-α-synuclein plasmids as previously described.11 Colocalization of α-synuclein (red signal) and synphilin-1 (green signal) was evident upon the superimposition of these two signals to produce a yellow color (third column Figure 2E). A “Colormap plugin” of ImageJ software was used to support (validate) the co-localization of synphilin-1 and α-synuclein proteins (extreme-right column Figure 2E).18,35 After 1 µM Aβ (2535) treatment for 24 h there a two-fold increase in the aggregation of synphilin-1 and α-synuclein was observed (when co-expressed) (P=0.0112 and P=0.0045, respectively) relative to the vehicle control (Figure 2F). Results from these experiments suggest that the increase in the aggregation of co-expressed synphilin-1 and α-synuclein is modulated by Aβ (25-35) presence (P=0.0218 and P=0.0083, respectively). SH-SY5Y cells containing aggregated Lewy body-like neurites (yellow arrow) were quantified to assess the impact of the Aβ (25-35) treatment (Supplementary Figure S1). Figure 2G clearly reveals the (adverse) effect of Aβ (25-35) on synphilin-1 and αsynuclein debris production [P=0.0239; 10 µM Aβ (25-35)]. There was ~ 4.0-fold increase relative to untreated condition in cells containing the toxic aggresomes upon 20 µM Aβ (25-35) treatment (P=0.0005). Collectively, these data confirm the cross-reactive capability of Aβ (2535) peptide fragment. We compared the expression levels of synphilin-1 and α-synuclein when these proteins were individually expressed or co-expressed in order to assess whether the expression of one protein influences the expression levels of the other (Figure 2H). Expression levels for synphilin8 ACS Paragon Plus Environment

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1 and α-synuclein are plotted in Figure 2H.i and 2H.ii, respectively. In both the cases, no difference in expression was found when these proteins where co-expressed but not treated with Aβ (25-35). Additionally, there was no significant change in α-synuclein expression when coexpressed with synphilin-1 and treated with Aβ (25-35), simultaneously; except, under conditions involving an included Aβ (25-35) concentration of 20 µM (P=0.0272). However, a significant increase in synphilin-1 expression was observed when co-expressed with α-synuclein and treated with Aβ (25-35) across all Aβ (25-35)concentrations tested (Figure 2H.i; P

An 11-mer Amyloid Beta Peptide Fragment Provokes Chemical

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