Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to ␣-synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in ␣-synuclein as indicators of metal-related conformational changes in ␣-synuclein, we show that iron and magnesium both interact with ␣-synuclein. ␣-Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of ␣-synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits ␣-synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases ␣-synuclein aggregation, while magnesium at concentrations >0.75 mM inhibits the aggregation of ␣-synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of ␣-synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation. Parkinson's disease (PD)1 is a common motor disorder that affects about 1% of population over the age of 65 (1). The disease is characterized by progressive neurodegeneration predominantly affecting dopaminergic neurons in the nigrostriatal system (2). The degenerating neurons develop intracellular inclusions, termed Lewy bodies, which are composed of a dense core of filamentous and granular material (3). Recent studies indicate that ␣-synuclein is a major filamentous component of Lewy bodies (3,4). Genetic studies suggest that ␣-synuclein plays a key role in the pathophysiology of PD, because mutations in ␣-synuclein, at A53T or A30P, are associated with early-onset familial PD (5, 6).The accumulation of aggregated protein underlies the pathophysiology of many neurodegenerative disorders, and increasing evidence suggests that aggregated ␣-synuclein plays a key role in the pathophysiology of PD. ␣-Synuclein has a strong tendency to aggregate and does so spontaneously in vitro at a slow rate (7-9). Both the A53T and the A30P mutations in PD increase the tendency of ␣-synuclein to aggregate. Many studies in cultured neurons, and some studies in transgenic animals, suggest that ␣-synuclein aggregation is linked to cellular toxicity and neurodegeneration (10 -12). In cell culture, formation of ␣-synuclein aggregates correlates with cell injury (10). Overexpressing ␣-synuclein in Drosophila leads to an age-dependent accumulation of aggregated ␣-synuclein and associated neurodegeneration (12). Masliah and colleagues also observed that aggregated ␣-synuclein is associated with loss of marke...

Multiple studies implicate iron in the pathophysiology of Parkinson's disease (PD). In the brains of patients with PD, iron levels are elevated and the levels of iron-binding proteins are abnormal. Iron has been suspected to contribute to PD because Fe(II) is known to promote oxidative damage. Recent studies suggest that an additional mechanism by which iron might contribute to PD is by inducing aggregation of the alpha-synuclein, which is a protein that accumulates in Lewy bodies in PD.

Parkin and alpha-synuclein are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Parkin is present in Lewy bodies and axonal spheroids in brains affected by PD, and mutations in parkin cause hereditary forms of Parkinsonism. Alpha-synuclein is a major component of Lewy bodies and is associated with rare cases of PD. We now show that parkin binds to alpha-synuclein, including conditions associated with alpha-synuclein aggregation. Parkin and alpha-synuclein complexes were observed in BE-M17 cells under basal conditions, in BE- M17 cells under oxidative conditions and in brains from control or PD donors. Double staining of PD brains shows parkin and alpha-synuclein co-localize to the same pathological structures (both Lewy bodies and axonal spheroids). These results suggest that parkin interacts with alpha-synuclein and could contribute to the pathophysiology of PD more generally than was previously considered.

Multicolor flow cytometric analysis is an invaluable tool for analyzing unique cell types within heterogeneous primary cell samples, like peripheral blood mononuclear cells (PBMCs). To identify these specific populations, antibodies against cell surface markers are typically used. However, obtaining greater in-depth knowledge often requires analysis of intracellular events, like phosphorylation of signaling molecules or expression of transcription factors. A challenge to staining cells for both cell surface and intracellular markers is the lack of compatibility of the cell fixation and permeabilization agents with the antibodies required for cell surface and intracellular staining. In this report, we developed an optimized protocol for the simultaneous detection of the cell signaling molecule Stat5 together with transcription factors and cell surface markers. We evaluated human PBMCs for phospho-Stat5 signal induction in Treg cells upon stimulation with IL-2. Using the optimized buffer protocol with antibodies at their optimal concentrations for this intracellular staining condition, we detected the phosphorylation of Stat5 in CD4+/CD25bright/FoxP3+ Treg cells in response to IL-2 stimulation. We also observed differential phosphorylation of Stat5 in various lymphocyte subsets, including memory and naive T cells, when human whole blood was stimulated with IL-2, lysed, fixed, permeabilized, and stained for CD3, CD4, CD45RA, CD45RO, T-bet, and pStat5 expression.

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Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, manufactured in a single test-per-tube, ready to use format. The dried backbone can be supplemented with liquid (drop in) reagents, which provides the flexibility of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and acquisition of millions of events is needed for data analysis. In addition, the dried reagent tubes have a long shelf life (12 months at 20–25°C vs. 24 hours for liquid cocktails with multiple BV dyes in BSB buffer at 2–8°C). Furthermore, utilization of multiple high-performing polymer dyes in the panel affords high resolution of the relevant cell populations. The dried plasma cell tube(s) were tested on bone marrow samples from healthy and diseased subjects in conjunction with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based CD38 and CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, and CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples (Kappa and Lambda).

Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely being used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, which can be supplemented with 2 liquid (drop in) reagents. Lyophilized or dried reagent cocktails in a unit test format are known to drive workflow efficiency in clinical settings by eliminating the need for manual cocktailing (of reagents), and frequent batch to batch verification procedures. Longer shelf life and room temperature storage are additional appealing features for lyophilized and dried products. We have developed a dried-down 8-color plasma cell panel, which is manufactured in a single test-per-tube, ready to use format, and can be supplemented with liquid (drop in) reagents. The flexibility of being able to add 2 liquid reagents to the dried backbone provides the option of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and there is a need for acquiring a large number of events for data analysis. Also, the dried reagent panel is comprised of multiple high performing polymer dyes, which are brighter than conventional dyes, and afford high resolution of the relevant cell populations. Furthermore, the dried reagent tubes have a long shelf life (12 months) and significantly enhance the workflow efficiency. Comparison of the liquid and dried versions of the 10-color plasma cell panel using peripheral blood from healthy donors (as well as control cell lines) showed similar staining patterns, comparable population statistics, and fluorochrome brightness. The dried plasma cell tube(s) were also tested on bone marrow samples from healthy and diseased subjects in conjugation with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based on CD38 & CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples. Citation Format: Ulrika Johansson, Na Li, Suraj Saksena, Natalie Golts, Brent Gaylord. Standardized approach for monitoring plasma cell disorders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4271.

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