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PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

ABSTRACT: Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 μm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH2) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.

SUBMITTER: Mazuryk J

PROVIDER: S-EPMC8200211 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

Mazuryk Jarosław J Puchalska Izabela I Koziński Kamil K Ślusarz Magdalena J MJ Ruczyński Jarosław J Rekowski Piotr P Rogujski Piotr P Płatek Rafał R Wiśniewska Marta Barbara MB Piotrowski Arkadiusz A Janus Łukasz Ł Skowron Piotr M PM Pikuła Michał M Sachadyn Paweł P Rodziewicz-Motowidło Sylwia S Czupryn Artur A Mucha Piotr P

International journal of molecular sciences 20210604 11

Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH2 (R49KKRRQRRR57-amide) and its less basic analogue, PTD4 (Y47ARAAARQARA57-amide). Our model included glucose deprivat ...[more]

PMID: 34200045

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Project description:Proper termination of cell-death-induced neural inflammation is the premise of tissue repair in acute ischemic stroke (AIS). Macrophages scavenge cell corpses/debris and produce inflammatory mediators that orchestrate immune responses. Here, we report that FOXP3, the key immune-repressive transcription factor of Tregs, is conditionally expressed in macrophages in stroke lesion. FOXP3 ablation in macrophages results in detrimental stroke outcomes, emphasizing the beneficial role of FOXP3+ macrophages. FOXP3+ macrophages are distinct from the M1 or M2 subsets and display superactive efferocytic capacity. With scRNAseq and analysis of FOXP3-bound-DNA isolated with CUT & RUN, we show that FOXP3 facilitates macrophage phagocytosis through enhancing cargo metabolism. FOXP3 expression is controlled by macroautophagic/autophagic protein degradation in resting macrophages, while initiation of LC3-associated phagocytosis (LAP) competitively occupies the autophagic machineries, and thus permits FOXP3 activation. Our data demonstrate a distinct set of FOXP3+ macrophages with enhanced scavenging capability, which could be a target in immunomodulatory therapy against AIS.Abbreviations: ADGRE1/F4/80: adhesion G protein-coupled receptor E1; AIF1/Iba1: allograft inflammatory factor 1; AIS: acute ischemic stroke; ARG1: arginase 1; ATP: adenosine triphosphate; BECN1/Beclin1: Beclin 1, autophagy related; BMDM: bone marrow-derived macrophages; CKO: conditional knockout; CSF1/M-CSF: colony stimulating factor 1 (macrophage); CSF2/GM-CSF: colony stimulating factor 2; CSF3/G-CSF: colony stimulating factor 3; CUT & RUN: cleavage under targets and release using nuclease; CyD: cytochalasin D; DAMP: danger/damage-associated molecular pattern; DIL: dioctadecyl-3,3,3,3-tetramethylin docarbocyanine; ELISA: enzyme linked immunosorbent assay; GO: Gene Ontology; FCGR3/CD16: Fc receptor, IgG, low affinity III; HMGB1: high mobility group box 1; IFNG/IFNγ: interferon gamma; IP: immunoprecipitation; KEGG: Kyoto Encyclopedia of Genes and Genomes; ITGAM/CD11b: integrin subunit alpha M; ITGAX/CD11c: integrin subunit alpha X; LAP: LC3-associated phagocytosis; LC-MS: liquid chromatography-mass spectrometry; LPS: lipopolysaccharide; MRC1/CD206: mannose receptor, C type 1; O4: oligodendrocyte marker O4; PBMC: peripheral blood mononuclear cells; RBC: red blood cells; PTPRC/CD45: protein tyrosine phosphatase, receptor type, C; RBFOX3/NeuN: RNA binding protein, fox 1 homolog (C. elegans) 3; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; scRNAseq: single cell RNA sequencing; SQSTM1/p62 (sequestosome 1); TGFB/TGFβ: transforming growth factor, beta; tMCAO: transient middle cerebral artery occlusion; TNF/TNFα: tumor necrosis factor; Treg: regulatory T cell.

Project description:BackgroundThe acute assessment of patients with suspected ischemic stroke remains challenging. The use of brain biomarker assays may improve the early diagnosis of ischemic stroke. The main goal of the study was to evaluate whether the NR2 peptide, a product of the proteolytic degradation of N-methyl-D-aspartate (NMDA) receptors, can differentiate acute ischemic stroke (IS) from stroke mimics and persons with vascular risk factors/healthy controls. A possible correlation between biomarker values and lesion sizes was investigated as the secondary objective.Methods and findingsA total of 192 patients with suspected stroke who presented within 72 h of symptom onset were prospectively enrolled. The final diagnosis was determined based on clinical observations and radiological findings. Additionally gender- and age-matched healthy controls (n = 52) and persons with controlled vascular risk factors (n = 48) were recruited to compare NR2 peptide levels. Blinded plasma was assayed by rapid magnetic particles (MP) ELISA for NR2 peptide within 30 min and results for different groups compared using univariate and multivariate statistical analyses. There was a clinical diagnosis of IS in 101 of 192 (53%) and non-stroke in 91 (47%) subjects. The non-stroke group included presented with acute stroke symptoms who had no stroke (n = 71) and stroke mimics (n = 20). The highest NR2 peptide elevations where found in patients with IS that peaked at 12 h following symptom onset. When the biomarker cut off was set at 1.0 ug/L, this resulted in a sensitivity of 92% and a specificity of 96% to detect IS. A moderate correlation (r(s) = 0.73) between NR2 peptide values and acute ischemic cortical lesions (<200 mL) was found.ConclusionsThis study suggests that the NR2 peptide may be a brain specific biomarker to diagnose acute IS and may allow the differentiation of IS from stroke mimics and controls. Additional larger scale clinical validation studies are required.

Project description:ObjectiveIn this study, we investigate whether a Convolutional Neural Network (CNN) can generate informative parametric maps from the pre-processed CT perfusion data in patients with acute ischemic stroke in a clinical setting.MethodsThe CNN training was performed on a subset of 100 pre-processed perfusion CT dataset, while 15 samples were kept for testing. All the data used for the training/testing of the network and for generating ground truth (GT) maps, using a state-of-the-art deconvolution algorithm, were previously pre-processed using a pipeline for motion correction and filtering. Threefold cross validation had been used to estimate the performance of the model on unseen data, reporting Mean Squared Error (MSE). Maps accuracy had been checked through manual segmentation of infarct core and total hypo-perfused regions on both CNN-derived and GT maps. Concordance among segmented lesions was assessed using the Dice Similarity Coefficient (DSC). Correlation and agreement among different perfusion analysis methods were evaluated using mean absolute volume differences, Pearson correlation coefficients, Bland-Altman analysis, and coefficient of repeatability across lesion volumes.ResultsThe MSE was very low for two out of three maps, and low in the remaining map, showing good generalizability. Mean Dice scores from two different raters and the GT maps ranged from 0.80 to 0.87. Inter-rater concordance was high, and a strong correlation was found between lesion volumes of CNN maps and GT maps (0.99, 0.98, respectively).ConclusionThe agreement between our CNN-based perfusion maps and the state-of-the-art deconvolution-algorithm perfusion analysis maps, highlights the potential of machine learning methods applied to perfusion analysis. CNN approaches can reduce the volume of data required by deconvolution algorithms to estimate the ischemic core, and thus might allow the development of novel perfusion protocols with lower radiation dose deployed to the patient.

Dataset Information

PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

Publications

PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke.

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