Cristine Betzer

How Epigallocatechin Gallate Can Inhibit α-Synuclein Oligomer Toxicity in Vitro

Background: Protein oligomers are implicated as cytotoxic membrane-disrupting agents in neurodegenerative diseases. Results: The small molecule EGCG, which inhibits α-synuclein oligomer toxicity, moderately reduces membrane binding and immobilizing the oligomer C-terminal tail. Conclusion: The α-synuclein oligomer may disrupt membranes by vesicle destabilization rather than pore formation. Significance: Limited reduction of oligomer membrane affinity may be sufficient to prevent cytotoxicity. Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCGs mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.

Characterizing the Dynamics of α-Synuclein Oligomers Using Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Soluble oligomers formed by α-synuclein (αSN) are suspected to play a central role in neuronal cell death during Parkinsons disease. While studies have probed the surface structure of these oligomers, little is known about the backbone dynamics of αSN when they form soluble oligomers. Using hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS), we have analyzed the structural dynamics of soluble αSN oligomers. The analyzed oligomers were metastable, slowly dissociating to monomers over a period of 21 days, after excess monomer had been removed. The C-terminal region of αSN (residues 94-140) underwent isotopic exchange very rapidly, demonstrating a highly dynamic region in the oligomeric state. Three regions (residues 4-17, 39-54, and 70-89) were strongly protected against isotopic exchange in the oligomers, indicating the presence of a stable hydrogen-bonded or solvent-shielded structure. The protected regions were interspersed by two somewhat more dynamic regions (residues 18-38 and 55-70). In the oligomeric state, the isotopic exchange pattern of the region of residues 35-95 of αSN corresponded well with previous nuclear magnetic resonance and electron paramagnetic resonance analyses performed on αSN fibrils and indicated a possible zipperlike maturation mechanism for αSN aggregates. We find the protected N-terminus (residues 4-17) to be of particular interest, as this region has previously been observed to be highly dynamic for both monomeric and fibrillar αSN. This region has mainly been described in relation to membrane binding of αSN, and structuring may be important in relation to disease.

Identification of Synaptosomal Proteins Binding to Monomeric and Oligomeric α-Synuclein

Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson’s disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.

Protein partners of α‐synuclein in Health and Disease

α‐synuclein is normally situated in the nerve terminal but it accumulates and aggregates in axons and cell bodies in synucleinopathies such as Parkinsons disease. The conformational changes occurring during α‐synucleins aggregation process affects its interactions with other proteins and its subcellular localization. This review focuses on interaction partners of α‐synuclein within different compartments of the cell with a focus on those preferentially binding aggregated α‐synuclein. The aggregation state of α‐synuclein also affects its catabolism and we hypothesize impaired macroautophagy is involved neuronal excretion of α‐synuclein species responsible for the prion‐like spreading of α‐synuclein pathology.

ELISA method to detect α-synuclein oligomers in cell and animal models

Soluble aggregates of α-synuclein, so-called oligomers, are hypothesized to act as neurotoxic species in Parkinson’s disease, Lewy body dementia and multiple systems atrophy, but specific tools to detect these aggregated species are only slowly appearing. We have developed an α-synuclein oligomer ELISA that allows us to detect and compare α-synuclein oligomer levels in different in vivo and in vitro experiments. The ELISA is based on commercially available antibodies and the epitope of the capture antibody MJF14-6-4-2 is folding- and aggregate-dependent and not present on monomers.

Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

Aggregation of α‐synuclein is a hallmark of Parkinsons disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α‐synuclein aggregation. Analyses of cell lines and primary culture models of α‐synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α‐synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+. CPA protects the cells against α‐synuclein‐aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo. Proximity ligation assays also reveal an increased interaction between α‐synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α‐synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down‐stream processes may be therapeutic targets for treating α‐synucleinopathies.

Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death

Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-β-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein.

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

Commentary: Alpha-Synuclein Aggregates Cause Calcium Dysregulation by Activating Calcium Pump SERCA

© 2018 Jensen PH. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License We have recently reported that alpha-synuclein aggregates induce calcium dysregulation by activating the calcium pump, SERCA in the endoplasmic reticulum1. The dysregulation presents as a biphasic change in cytosolic calcium with an initial phase with 20% decreased calcium followed by final phase with increased calcium. These findings are novel with respect to; 1) demonstrating activation of SERCA as a gain-offunction acquired by aggregated alpha-synuclein; 2) demonstrating an initial phase with reduced cytosolic calcium in neurons that experience a build-up of alpha-synuclein aggregates. The initial phase occurs when cells appear morphologically unaffected and precedes the wellknown later phase with increased cytosolic calcium and subsequent cell death. Although the reduction in cytosolic calcium of 20% may appear modest are its short and long-term consequences largely unknown because this phase not previously has been described. However, both the early and late phase of the calcium dysregulation can be antagonized pharmacologically by inhibiting SERCA and this treatment restored the survival of the cells to the level of the control cells not being subject to alpha-synuclein aggregate stress. The interaction between aggregated synuclein and SERCA could be demonstrated in neurons in brain tissue affected by dementia with Lewy bodies supporting that the initial phase with reduced cytosolic calcium is relevant for human pathology. We hypothesize the alpha-synuclein aggregate induced stimulation of SERCA and the early-phase with reduced cytosolic calcium will compromise neurons ability to support their role in normal circuitries, integrated brain functions and ultimately cause symptoms. This phase may exist for prolonged periods before the neurons enters the latephase with increased cytosolic calcium that has been studied according to the original “calcium hypothesis”2,3. Understanding the mechanisms whereby the abnormal activation of SERCA and its down-stream dysregulation of calcium contributes to neuronal dysfunction and eventual cell death holds potential for new symptomatic and disease modifying strategies for synucleinopathies.