Decreased levels of the gelsolin plasma isoform in patients with rheumatoid arthritis

Introduction Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of $200 \pm 50$ mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis.Methods Circulating and intra-articular levels of plasma gelsolin were measured in 78 patients with rheumatoid arthritis using a functional (pyrene-actin nucleation) assay and compared with 62 age- and gender-matched healthy controls.Results Circulating plasma gelsolin levels were significantly lower in patients with rheumatoid arthritis compared with healthy controls ($141 \pm 32$ versus $196 \pm 40$ mg/L, P = 0.0002). The patients' intra-articular plasma gelsolin levels were significantly lower than in the paired plasma samples ($94 \pm 24$ versus $141 \pm 32$ mg/L, P = 0.0001). Actin was detected in the synovial fluids of all but four of the patients, and immunoprecipitation experiments identified gelsolin-actin complexes.Conclusions The plasma isoform of gelsolin is decreased in the plasma of patients with rheumatoid arthritis compared with healthy controls. The reduced plasma concentrations in combination with the presence of actin and gelsolin-actin complexes in synovial fluids suggest a local consumption of this potentially anti-inflammatory protein in the inflamed joint

Comprehensive Cell Surface Antigen Analysis Identifies Transferrin Receptor Protein-1 (CD71) as a Negative Selection Marker for Human Neuronal Cells.

Cell state-, developmental stage-, and lineage-specific combinatorial expression of cluster of differentiation (CD) molecules enables the identification of cellular subsets via multicolor flow cytometry. We describe an exhaustive characterization of neural cell types by surface antigens, exploiting human pluripotent stem cell-derived neural cell systems. Using multiwell screening approaches followed by detailed validation of expression patterns and dynamics, we exemplify a strategy for resolving cellular heterogeneity in stem cell paradigms. In addition to providing a catalog of surface antigens expressed in the neural lineage, we identified the transferrin receptor-1 (CD71) to be differentially expressed in neural stem cells and differentiated neurons. In this context, we describe a role for N-Myc proto-oncogene (MYCN) in maintaining CD71 expression in proliferating neural cells. We report that in vitro human stem cell-derived neurons lack CD71 surface expression and that the observed differential expression can be used to identify and enrich CD71- neuronal derivatives from heterogeneous cultures. Stem Cells 2019;37:1293-1306

Molecular basis of filamin a-filGAP interaction and its impairment in congenital disorders associated with filamin a mutations

Peer reviewe

Mechanism of F-actin crosslinking by filamin A and the anti-inflammatory functions of plasma gelsolin in bodily fluids

Gelsolin (GSN) and filamin A (FLNa) are two actin-binding proteins discovered in our laboratory over 30 years ago. GSN is a calcium-activated actin severing and barbed end capping protein that is expressed as both intracellular and extracellular (plasma gelsolin, pGSN) isoforms. pGSN is present at relatively high concentrations (~ 200 µg/ml) in blood, but its extracellular functions have not been determined. pGSN levels decrease during acute inflammation and low levels correlate negatively with survival. Re-administration of pGSN to severely injured animals can rescue them from death, although the mechanism for this is unknown. pGSN levels during chronic inflammation have not been reported. FLNa is an important architectural component of three-dimensional actin networks in cells. It is an elongated homo-dimer that efficiently crosslinks F-actin into a gel in contrast to the gel-solating properties of GSN. Each subunit has an N-terminal “actin-binding domain” (ABD) followed by two rod-like domains and a C-terminal self-association domain. FLNa mediates actin-membrane connections, serves as a scaffold for >50 different binding partners, and FLNa-F-actin crosslinks accommodate cell shape changes and motility. However, as of yet there have not been sufficient details concerning FLNa’s structure to fully explain its multiplicity of functions. pGSN has lipid-binding sites and has been shown to bind to lysophosphatidic acid (LPA), a potent cell-activating phospholipid. Based on this, a new hypothesis positing pGSN as an anti-inflammatory protein was formed. Using platelets and neutrophils isolated from human blood, the effects of recombinant pGSN on platelet P-selectin exposure and neutrophil oxygen radical production induced by LPA and another structurally related phospholipid, platelet-activating factor (PAF), were investigated. Results showed that pGSN modulated cellular activation induced by both of these inflammatory phospholipids. In order to investigate pGSN levels during chronic inflammation, plasma and synovial fluids from patients with rheumatoid arthritis were analyzed. pGSN levels were lower in plasma from patients than age and gender matched healthy controls, and further reduced in synovial fluid. To examine the mechanism behind FLNa’s potency as a F-actin crosslinker, the FLNa-F-actin interaction was investigated by binding and gel-point assays, electron microscopy, and real-time video microscopy using full-length and truncated FLNa molecules. A new F-actin binding site was identified, which functions in conjunction with dimerization, long flexible subunits, and the previously identified ABD, to explain high avidity binding to F-actin. The results also show that crosslinks are rigid structures and that the self-association domains determine high angle branching. The C-T domain of FLNa, which binds many partners, has a compact structure compared to the elongated N-T two-thirds of the protein, does not associate with F-actin and can bind partners while FLNa is bound to F-actin. In conclusion, these findings demonstrate a novel function of pGSN as a modulator of phospholipids, a finding that may be important for inflammation, and that pGSN levels are decreased during chronic inflammation in addition to previously documented acute conditions. The mechanism of FLNa crosslinking of F-actin can be explained by the intrinsic structure and properties of the FLNa molecule

A Nurr1 Agonist Causes Neuroprotection in a Parkinson’s Disease Lesion Model Primed with the Toll-Like Receptor 3 dsRNA Inflammatory Stimulant Poly(I:C)

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) are characterized by the expression of genes required for dopamine synthesis, handling and reuptake and the expression of these genes is largely controlled by nuclear receptor related 1 (Nurr1). Nurr1 is also expressed in astrocytes and microglia where it functions to mitigate the release of proinflammatory cytokines and neurotoxic factors. Given that Parkinson’s disease (PD) pathogenesis has been linked to both loss of Nurr1 expression in the SNpc and inflammation, increasing levels of Nurr1 maybe a promising therapeutic strategy. In this study a novel Nurr1 agonist, SA00025, was tested for both its efficiency to induce the transcription of dopaminergic target genes in vivo and prevent dopaminergic neuron degeneration in an inflammation exacerbated 6-OHDA-lesion model of PD. SA00025 (30mg/kg p.o.) entered the brain and modulated the expression of the dopaminergic phenotype genes TH, VMAT, DAT, AADC and the GDNF receptor gene c-Ret in the SN of naive rats. Daily gavage treatment with SA00025 (30mg/kg) for 32 days also induced partial neuroprotection of dopaminergic neurons and fibers in rats administered a priming injection of polyinosinic-polycytidylic acid (poly(I:C) and subsequent injection of 6-OHDA. The neuroprotective effects of SA00025 in this dopamine neuron degeneration model were associated with changes in microglial morphology indicative of a resting state and a decrease in microglial specific IBA-1 staining intensity in the SNpc. Astrocyte specific GFAP staining intensity and IL-6 levels were also reduced. We conclude that Nurr1 agonist treatment causes neuroprotective and anti-inflammatory effects in an inflammation exacerbated 6-OHDA lesion model of PD

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons

Abstract The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM+/CD29low sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM+/CD29low DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM+/CD29low DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.</jats:p