BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy ...receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-xL positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.
Background: Bnip3 is both a pro-apoptotic BH3-only protein and a mitochondrial autophagy receptor.
Results: Serine phosphorylation of the Bnip3 LC3-interacting region (LIR) increased binding to Atg8 members and consequently mitophagy, in a manner positively regulated by Bcl-xL.
Conclusion: The Bnip3 LIR activity state determines either pro-survival mitophagy or mitochondrial apoptosis.
Significance: Bnip3-induced mitophagy is serine kinase-regulated and thus a targetable pathway.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the ...Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in ...phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function.
We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays.
PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function.
The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human ...brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis.
Full text
Available for:
IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Antiapoptotic Bcl-2 family members suppress both apoptosis and autophagy and are of major importance for therapy resistance of malignant gliomas. To target these molecules, we used BH3 mimetics and ...analyzed the molecular mechanisms of cell death induced thereby. Glioma cells displayed only limited sensitivity to single-agent treatment with the BH3 mimetics HA14-1, BH3I-2', and ABT-737, whereas the pan-Bcl-2 inhibitor (-)-gossypol efficiently induced cell death. Furthermore, (-)-gossypol potentiated cell death induced by temozolomide (TMZ) in MGMT (O(6)-methylguanine-DNA methyltransferase)-negative U343 cells and, to a lesser extent, in MGMT-expressing U87 cells. (-)-Gossypol triggered translocation of light chain 3 to autophagosomes and lysosomes and cytochrome c release, but cell death occurred in the absence of lysosomal damage and effector caspase activation. Lentiviral knockdown of Beclin1 and Atg5 in U87, U343, and MZ-54 cells strongly diminished the extent of cell death induced by (-)-gossypol and combined treatment with TMZ, indicating that autophagy contributed to this type of cell death. In contrast, stable knockdown of the endogenous autophagy inhibitor mammalian target of rapamycin increased autophagic cell death. Our data suggest that pan-Bcl-2 inhibitors are promising drugs that induce caspase-independent, autophagic cell death in apoptosis-resistant malignant glioma cells and augment the action of TMZ. Furthermore, they indicate that efficient killing of glioma cells requires neutralization of Mcl-1.
Soils provide many functions as they represent a habitat for flora and fauna, supply water, nutrient, and anchorage for plant growth and more. They can also be considered as large bioreactors in ...which many processes occur that involve the consumption and production of different gas species. Soils can be a source and sink for greenhouse gases. During the last decades this topic attracted special attention. Most studies on soil-atmosphere gas fluxes used chamber methods or micro-meteorological methods. Soil gas fluxes can also be calculated from vertical soil gas profiles which can provide additional insights into the underlying processes. We present a design for sampling and measuring soil gas concentration profiles that was developed to facilitate long term monitoring. Long term monitoring requires minimization of the impact of repeated measurements on the plot and also minimization of the routine workload while the quality of the measurement needs to be maintained continuously high. We used permanently installed gas wells that allowed passive gas sampling at different depths. Soil gas monitoring set ups were installed on 13 plots at 6 forest sites in South West Germany between 1998 and 2010. Until now, soil gas was sampled monthly and analysed for CO2, N2O, CH4, O2, N2, Ar, and C2H4 using gas chromatography. We present typical time series and profiles of soil gas concentrations and fluxes of a selected site as an example. We discuss the effect of different calculation approaches and conclude that flux estimates of O2, CO2 and CH4 can be considered as highly reliable, whereas N2O flux estimates include a higher uncertainty. We point out the potential of the data and suggest ideas for future research questions for which soil gas monitoring would provide the ideal data basis. Combining and linking the soil gas data with additional environmental data promises new insights and understanding of soil processes.
In a previous study, Protein Kinase C iota (PRKCI) emerged as an important candidate gene for glioblastoma (GBM) stem‐like cell (GSC) survival. Here, we show that PKCι is overexpressed and activated ...in patient derived GSCs compared with normal neural stem cells and normal brain lysate, and that silencing of PRKCI in GSCs causes apoptosis, along with loss of clonogenicity and reduced proliferation. Notably, PRKCI silencing reduces tumor growth in vivo in a xenograft mouse model. PKCι has been intensively studied as a therapeutic target in non‐small cell lung cancer, resulting in the identification of an inhibitor, aurothiomalate (ATM), which disrupts the PKCι/ERK signaling axis. However, we show that, although sensitive to pharmacological inhibition via a pseudosubstrate peptide inhibitor, GSCs are much less sensitive to ATM, suggesting that PKCι acts along a different signaling axis in GSCs. Gene expression profiling of PRKCI‐silenced GSCs revealed a novel role of the Notch signaling pathway in PKCι mediated GSC survival. A proximity ligation assay showed that Notch1 and PKCι are in close proximity in GSCs. Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM.
What's new?
Increased understanding of gene expression patterns in glioblastoma (GBM) has led to the identification of genes involved in survival, among them protein kinase C iota (PRKCI). PRKCI is classified as an oncogene in several human cancer types. In this study, PRKCI was found to be overexpressed in patient‐derived GBM stem‐like cells (GSCs) and to play a crucial role in GSC survival through Notch signaling. Its silencing slowed tumor growth and prolonged survival in a xenograft GBM mouse model. The findings highlight the therapeutic promise of PKCι and its potential to provide a new avenue for GSC‐targeted GBM therapies.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Introduction
Obsessive-compulsive disorder (OCD) affects 2–3% of the global population, causing distress in many functioning levels. Standard treatments only lead to a partial recovery, and about 10% ...of the patients remain treatment-resistant. Deep brain stimulation offers a treatment option for severe, therapy-refractory OCD, with a reported response of about 60%. We report a comprehensive clinical, demographic, and treatment data for patients who were treated with DBS in our institution.
Methods
We offered DBS to patients with severe chronic treatment resistant OCD. Severity was defined as marked impairment in functioning and treatment resistance was defined as non-response to adequate trials of medications and psychotherapy. Between 2020 and 2022, 11 patients were implanted bilaterally in the bed nucleus of stria terminalis (BNST). Patients were evaluated with YBOCS, MADRS, GAF, CGI, and WHOQOL-BREF. We performed the ratings at baseline (before surgery), after implantation before the start of the stimulation, after reaching satisfactory stimulation parameters, and at follow-up visits 3, 6, 9, and 12 months after optimized stimulation.
Results
One patient has retracted his consent to publish the results of his treatment, thus we are reporting the results of 10 patients (5 males, 5 females, mean age: 37 years). Out of our 10 patients, 6 have shown a clear response indicated by a YBOCS-reduction between 42 and 100 percent at last follow-up. One further patient experienced a subjectively dramatic effect on OCD symptoms, but opted afterwards to stop the stimulation. The other 3 patients showed a slight, non-significant improvement of YBOCS between 8.8 and 21.9%. The overall mean YBOCS decreased from 28.3 at baseline to 13.3 (53% reduction) at the last follow-up. The improvement of the OCD symptoms was also accompanied by an improvement of depressive symptoms, global functioning, and quality of life.
Conclusion
Our results suggest that BNST-DBS can be effective for treatment-resistant OCD patients, as indicated by a reduction in symptoms and an overall improvement in functioning. Despite the need for additional research to define the patients’ selection criteria, the most appropriate anatomical target, and the most effective stimulation parameters, improved patient access for this therapy should be established.
Background
Forest liming is a common practice in many German forests, which aims primarily at improving soil chemical properties that have been negatively impacted by acid rain. Effects on physical ...functions have not yet been studied widely.
Aims
The hypothesis of this study is that chemical changes after liming lead to changes in the physical properties of the humus layer and mineral soil.
Methods
We studied soil physical characteristics (water retention characteristics, air and water permeabilities, and conductivities) of limed and adjacent control plots of sandy to loamy, acidified soils under spruce and beech‐dominated stands.
Results
We found differences between limed and control plots, especially in the humus layer and mineral top soil. Here, air capacity values (representing air‐filled macropores at −6 kPa) have experienced an increase, while available water capacities (representing the sum of dewatered mesopores between −6 and −1500 kPa) were reduced to a variable extent. These changes in pore size distributions affected gas diffusion as well as gas permeability positively. Unsaturated hydraulic conductivity was not affected. Below the top mineral soil, a tendency to a reduced macroporosity and gas permeability was observed, but no clear changes were detected.
Conclusions
Small but significant changes of soil physical properties and functions in the humus layer and top mineral soil are discussed to be an indirect effect of forest liming. Liming improves pH and nutrient availability, which in turn should have stimulated activity and abundance of soil fauna, especially earthworms. Where observed, their activity could explain the observed changes in soil physical properties. We believe that effects below the top soil might be due to mobilization and translocation of soil particles and a clogging of pores, but these depths were only studied at three plots. Compared to common liming practices, however, the investigated sites are characterized by significantly higher lime applications. The effects on practice liming areas in forestry praxis are therefore likely to be smaller than in this study.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Due to the rapid microbial degradation, ethylene (ethene; C
2
H
4
) detection in most soils is difficult. The knowledge about factors favoring the occurrence of C
2
H
4
is mainly based on laboratory ...experiments which are not necessarily representative for real field conditions in forest soils. We report results from a total of 24 measuring plots in southwest Germany and more than 50,000 gas samples, including long-term forest monitoring sites and other study sites involving liming, artificial soil compaction, and temporary waterlogging. Many of the patterns and influencing factors identified in the laboratory were confirmed. In well-aerated forest soils, C
2
H
4
was detected in 1.3% of the gas samples. C
2
H
4
detection was higher at oxygen (O
2
) concentrations below 10% in the soil. The effect was positively correlated with bulk density in compacted soils and CO
2
, CH
4
, and N
2
O concentrations—factors that indicate reduced microbial activity and thus also facilitate the detection of C
2
H
4
. It could be detected 3.5 times more often in spruce than in beech stands, indicating that plant species plays a role and was not found to follow any distinct spatial or temporal pattern with the exception of seemingly random spatio-temporal clusters of root-born C
2
H
4
near large roots of spruce trees. The long observation period and large dataset of this field study allowed valuable insights into C
2
H
4
occurrence in forest soils under natural conditions supporting the theory that the plant hormone C
2
H
4
is produced as a warning for anaerobic soil areas that are limiting root growth.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
PDF
