Follicular dendritic cells emerge from ubiquitous perivascular precursors

The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ(+)-derived cells abolished FDC, indicating that FDC originate from PDGFRβ(+) cells. Lymphotoxin-α-overexpressing prion protein (PrP)(+) kidneys developed PrP(+) FDC after transplantation into PrP(-) mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ(+) stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR)(-) kidney capsules, differentiated into Mfge8(+)CD21/35(+)FcγRIIβ(+)PrP(+) FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ(+) FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.     

Temperature impact on the midsummer decline of Daphnia galeata: an analysis of long-term data from the biomanipulated Bautzen Reservoir (Germany)

1. The influence of water temperature on occurrence and duration of a midsummer decline (MSD) of Daphnia galeata was studied in the biomanipulated Bautzen Reservoir in Germany. The proportion of piscivores in the fish community of the reservoir has been enhanced experimentally since 1981. As a consequence, Daphnia galeata has dominated the zooplankton. Over 18 years of study (1981–1998), a long‐lasting MSD (longer than 30 days) occurred in 7 years, whereas a short MSD (shorter than 30 days) was observed in 6 years. During the remaining 5 years, an MSD was not observed.
2. Two hypotheses were examined to explain the observed patterns. First, we postulated that high water temperature during winter and early spring (January–April) leads to an MSD after an early and high spring peak of daphnids. On the other hand, low temperature during winter and early spring should not cause an MSD owing to a slower increase of the population, resulting in a later peak of daphnids. Second, we hypothesized that the mean water temperature during early summer (May and June) influences the occurrence of an MSD (by controlling young‐of‐the‐year (YOY) fish predation on daphnids).
3. The water temperature during winter and early spring explains 83%, and the early summer water temperature 55%, of interannual variation in the occurrence of an MSD.
4. The interannual variation in duration of an MSD was neither explained by temperature during winter and early spring nor by early summer temperature alone, but in 14 of the 18 years (78%) by a combination of both.
5. We conclude that water temperature during winter and early spring had a strong impact on Daphnia mortality by influencing height and timing of the spring peak which, in turn, influenced the extent of overexploitation of their food resources. By contrast, the water temperature during early summer probably influenced the mortality of daphnids caused by predation of YOY fish. The relative timing of both sources of mortality, which depends on the temperature regime during the first 6 months of the year, is the key process in controlling the occurrence and duration of an MSD. A long‐lasting MSD, therefore, is likely in Bautzen Reservoir only if temperatures are high during winter and early spring, as well as during early summer.
6. As a consequence of climate warming, recent climate records reveal warming during winter, spring and early summer in middle Europe, rather than an increase in mean annual temperatures. If our findings and conclusions are related to this regional and temporal pattern of climate warming, an increasing frequency of years with a long‐lasting MSD and, consequently, a decreasing efficiency of biomanipulation can be predicted.     

The γ-Glutamyl Transpeptidase Inhibitor Acivicin Preserves Glutathione Released by Astroglial Cells in Culture

The release of glutathione from astroglial cells was investigated using astroglia-rich primary cultures prepared from the brains of newborn rats. These cells release glutathione after onset of an incubation in a glucose-containing minimal medium. The amount of extracellular glutathione increased with the time of incubation, although the accumulation slowed down gradually. An elevated rate of increase of the glutathione concentration in the incubation medium was found if the astroglial ectoenzyme -glutamyl transpeptidase was inhibited by acivicin. The activity of -glutamyl transpeptidase in astroglia-rich primary cultures, which was found to be 1.9 ± 0.3 nmol/(min × mg protein), was markedly reduced if the cells had been incubated in the presence of acivicin. After 2 h of incubation with acivicin half-maximal and maximal inhibition of -glutamyl transpeptidase activity was found at concentrations of about 5 M and 50 M, respectively. In the presence of acivicin at a concentration above 10 M the glutathione content found released from astroglial cells apparently increased almost proportional to time for up to 10 h. Under these conditions the average rate of release was 2.1 ± 0.3 nmol/(h × mg protein) yielding after a 10 h incubation an extracellular glutathione content three times that of the medium of cells incubated without inhibitor. Half-maximal and maximal effects on the level of extracellular glutathione were found at 4 M and 50 M acivicin, respectively. After a 10 h incubation with acivicin the intracellular content of glutathione was reduced to 75% of the level of untreated astroglial cultures. These results suggest that glutathione released from astroglial cells can serve as substrate for the ectoenzyme -glutamyl transpeptidase of these cells.     

VEGFR-3/Flt-4 mediates proliferation and chemotaxis in glial precursor cells

Neuronal and vascular cells share common chemical signals. Vascular endothelial growth factor (VEGF)-C and -D and their receptor VEGFR-3/Flt-4 mediate lymphangiogenesis, but they occur also in the brain. Quantitative RT-PCR of mouse brain tissues and cultivated cells showed that the VEGFR-3 gene is highest transcribed in postnatal brain and in glial precursor cells whereas VEGF-C and -D are variably produced by different neuronal and glial cells. In neurospheres (neural stem cells) VEGFR-3 was induced by differentiation with platelet-derived growth factor (PDGF). In functional studies with an A2B5- and nestin-positive, O4-negative murine glial precursor cell line, VEGF-C and -D stimulated phosphorylation of the kinases Erk1/2; this signal transduction was inhibited by UO126. Both peptides induced the proliferation of glial precursor cells which could be inhibited by UO126. Furthermore, VEGF-D considerably enhanced their migration into an open space in a wound-healing assay. These results show that VEGF-C/-D together with its receptor VEGFR-3 provides an auto-/paracrine growth and chemotactic system for glial precursors in the developing brain.     

Impaired function and delayed regeneration of dendritic cells in COVID-19

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage⁻HLADR⁺ cells lacking DC markers. Increased frequency of CD163⁺ CD14⁺ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4⁺ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.     

Surface-enhanced vibrational spectroscopy for probing transient interactions of proteins with biomimetic interfaces: electric field effects on structure, dynamics and function of cytochrome c

Most of the biochemical and biophysical processes of proteins take place at membranes, and are thus under the influence of strong local electric fields, which are likely to affect the structure as well as the reaction mechanism and dynamics. To analyse such electric field effects, biomimetic interfaces may be employed that consist of membrane models deposited on nanostructured metal electrodes. For such devices, surface-enhanced resonance Raman and IR absorption spectroscopy are powerful techniques to disentangle the complex interfacial processes of proteins in terms of rotational diffusion, electron transfer, and protein and cofactor structural changes. The present article reviews the results obtained for the haem protein cytochrome c, which is widely used as a model protein for studying the various reaction steps of interfacial redox processes in general. In addition, it is shown that electric field effects may be functional for the natural redox processes of cytochrome c in the respiratory chain, as well as for the switch from the redox to the peroxidase function, one of the key events preceding apoptosis.     

Exosomal cell-to-cell transmission of alpha synuclein oligomers

ABSTRACT: BACKGROUND: Aggregation of alpha-synuclein (alphasyn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson's disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of alphasyn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted alphasyn across membranes. The aim of this study is to characterize the transcellular spread of alphasyn oligomers and determine their extracellular location. RESULTS: Using a novel protein fragment complementation assay where alphasyn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that alphasyn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated alphasyn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free alphasyn oligomers. Specifically, we determine that alphasyn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of alphasyn oligomers is strongly influenced by autophagic activity. CONCLUSIONS: Our data suggest that alphasyn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of alphasyn oligomers is a mechanism whereby cells clear toxic alphasyn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in alphasyn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies.     

Exosomal cell-to-cell transmission of alpha synuclein oligomers

Background

Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF.

Results

NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-?? signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A.

Conclusions

Our results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.     

Liposome-siRNA-Peptide Complexes Cross the Blood-Brain Barrier and Significantly Decrease PrPC on Neuronal Cells and PrPRES in Infected Cell Cultures

Background

Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrP^C expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrP^C expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo.

Methodology/Principal Findings

Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs), acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation.

Conclusions/Significance

Liposome-siRNA-peptide complexes (LSPCs) delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrP^C expression and eliminated PrP^RES formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrP^C-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.