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.

Increased motor neuron resilience by small molecule compounds that regulate IGF-II expression.

The selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is evident by sparing of a few subpopulations during this fast progressing and debilitating degenerative disease. By studying the gene expression profile of resilient vs. vulnerable motor neuron populations we can gain insight in what biomolecules and pathways may contribute to the resilience and vulnerability. Several genes have been found to be differentially expressed in the vulnerable motor neurons of the cervical spinal cord as compared to the spared motor neurons in CNIII/IV. One gene that is differentially expressed and present at higher levels in less vulnerable motor neurons is insulin-like growth factor II (IGF-II). The motor neuron protective effect of IGF-II has been demonstrated both in vitro and in SOD1 transgenic mice. Here, we have screened a library of small molecule compounds and identified inducers of IGF-II mRNA and protein expression. Several identified compounds significantly protected motor neurons from glutamate excitotoxicity in vitro. One of the compounds, vardenafil, resulted in a complete motor neuron protection, an effect that was reversed by blocking receptors of IGF-II. When administered to naïve rats vardenafil was present in the cerebrospinal fluid and increased IGF-II mRNA expression in the spinal cord. When administered to SOD1 transgenic mice, there was a significant delay in motor symptom onset and prolonged survival. Vardenafil also increased IGF-II mRNA and protein levels in motor neurons derived from healthy subject and ALS patient iPSCs, activated a human IGF-II promoter and improved survival of ALS-patient derived motor neurons in culture. Our findings suggest that modulation of genes differentially expressed in vulnerable and resilient motor neurons may be a useful therapeutic approach for motor neuron disease.

Direct dynamin-actin interactions regulate the actin cytoskeleton.

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.

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.

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(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) 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(+) /CD29(low) 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.

Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.

Development of histocompatible primate-induced pluripotent stem cells for neural transplantation.

Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. To develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CMs by microsatellite polymerase chain reaction analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson's disease and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to 6 months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the preclinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.

Global gene expression profiling of somatic motor neuron populations with different vulnerability identify molecules and pathways of degeneration and protection.

Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration.

Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.

The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/ß-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.

Advantages and Recent Developments of Autologous Cell Therapy for Parkinson's Disease Patients.

Parkinson's Disease (PD) is a progressive degenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. There are approximately 7-10 million PD patients worldwide. Currently, there are no biomarkers available or pharmaceuticals that can halt the dopaminergic neuron degeneration. At the time of diagnosis about 60% of the midbrain dopamine (mDA) neurons have already degenerated, resulting in a depletion of roughly 70% of striatal dopamine (DA) levels and synapses. Symptomatic treatment (e.g., with L-dopa) can initially restore DA levels and motor function, but with time often lead to side-effects like dyskinesia. Deep-brain-stimulation can alleviate these side-effects and some of the motor symptoms but requires repeat procedures and adds limitations for the patients. Restoration of dopaminergic synapses using neuronal cell replacement therapy has shown benefit in clinical studies using cells from fetal ventral midbrain. This approach, if done correctly, increases DA levels and restores synapses, allowing biofeedback regulation between the grafted cells and the host brain. Drawbacks are that it is not scalable for a large patient population and the patients require immunosuppression. Stem cells differentiated in vitro to mDA neurons or progenitors have shown promise in animal studies and is a scalable approach that allows for cryopreservation of transplantable cells and rigorous quality control prior to transplantation. However, all allogeneic grafts require immunosuppression. HLA-donor-matching, reduces, but does not completely eliminate, the need for immunosuppression, and is currently investigated in a clinical trial for PD in Japan. Since immune compatibility is very important in all areas of transplantation, these approaches may ultimately be of less benefit to the patients than an autologous approach. By using the patient's own somatic cells, reprogrammed to induced pluripotent stem cells (iPSCs) and differentiated to mDA neurons immunosuppression is not required, and may also present with several biological and functional advantages in the patients, as described in this article. The proof-of-principle of autologous iPSC mDA restoration of function has been shown in parkinsonian non-human primates (NHPs), and this can now be investigated in clinical trials in addition to the allogeneic and HLA-matched approaches. In this review, we focus on the autologous approach of cell therapy for PD.

Neurite Collapse and Altered ER Ca2+ Control in Human Parkinson Disease Patient iPSC-Derived Neurons with LRRK2 G2019S Mutation.

The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.

Seq-ing Markers of Midbrain Dopamine Neurons.

Transplantation of human pluripotent stem cell-derived dopaminergic neurons is a promising approach to treating Parkinson's disease. In this issue of Cell Stem Cell, Kee et al. (2017) and Kirkeby et al. (2017) identify specific markers of midbrain dopaminergic progenitors to improve their derivation and predict dopamine neuron content after engraftment.

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.

Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease.

Autologous transplantation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility, and safety of iPSC-derived dopamine neurons in non-human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinson's disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10µM), or large inclusions (≥10µM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.

Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q(10), rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.

Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

BACKGROUND: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. METHODOLOGY/PRINCIPAL FINDINGS: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress. CONCLUSIONS/SIGNIFICANCE: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

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 +/- 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 +/- 32 versus 196 +/- 40 mg/L, P = 0.0002). The patients' intra-articular plasma gelsolin levels were significantly lower than in the paired plasma samples (94 +/- 24 versus 141 +/- 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.

Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin.

Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform, plasma gelsolin (pGSN). Blood concentrations of pGSN decrease in response to diverse tissue injuries. Depletion of pGSN to critical levels precedes and often predicts complications of injuries such as lung permeability changes and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. One proposed mechanism for pGSN's protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. However, no direct evidence in support of this hypothesis has been available. Here we show that recombinant pGSN modestly inhibited LPA-induced P-selectin upregulation by human platelets in the presence of albumin (P < 0.0001). However, physiologically relevant pGSN concentrations inhibit platelet-activating factor (PAF)-mediated P-selectin expression by up to 77% (P < 0.0001). pGSN also markedly inhibited PAF-induced superoxide anion (O(2)(-)) production of human peripheral neutrophils (PMN) in a concentration-dependent manner (P < 0.0001). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O(2)(-) generation (P = 0.024). Therefore, pGSN interferes with PAF- and LPA-induced cellular activation in vitro, suggesting a mechanism for the protective role of pGSN in vivo.

Structural basis of filamin A functions.

Filamin A (FLNa) can effect orthogonal branching of F-actin and bind many cellular constituents. FLNa dimeric subunits have N-terminal spectrin family F-actin binding domains (ABDs) and an elongated flexible segment of 24 immunoglobulin (Ig) repeats. We generated a library of FLNa fragments to examine their F-actin binding to define the structural properties of FLNa that enable its various functions. We find that Ig repeats 9-15 contain an F-actin-binding domain necessary for high avidity F-actin binding. Ig repeats 16-24, where most FLNa-binding partners interact, do not bind F-actin, and thus F-actin does not compete with Ig repeat 23 ligand, FilGAP. Ig repeats 16-24 have a compact structure that suggests their unfolding may accommodate pre-stress-mediated stiffening of F-actin networks, partner binding, mechanosensing, and mechanoprotection properties of FLNa. Our results also establish the orientation of FLNa dimers in F-actin branching. Dimerization, mediated by FLNa Ig repeat 24, accounts for rigid high-angle FLNa/F-actin branching resistant to bending by thermal forces, and high avidity F-actin binding and cross-linking.