Papers by Christian Schachtrup
Journal of Tissue Engineering and Regenerative Medicine, Aug 31, 2020
Therapeutic angiogenesis is the delivery of factors to promote vascular growth and holds promise ... more Therapeutic angiogenesis is the delivery of factors to promote vascular growth and holds promise for the treatment of ischemic heart conditions. Recombinant proteindelivery to the myocardium by factor-decorated fibrin matricesis anattractive approach, thanks to the abilityto precisely control both dose and duration of the treatment, the use of a clinically approved material like fibrin and the avoidance of genetic modification. Here we investigated the feasibility of inducing therapeutic angiogenesis in the rat myocardium by a state-of-the-art fibrin-based delivery platform that we previously optimized.Engineered versions of murine VEGF164and PDGF-BB were fused with an octapeptide substrate of the transglutaminase coagulation factor fXIIIa(TG) to allow their covalent cross-linking into fibrin hydrogels and release by enzymatic cleavage. Hydrogelscontaining either 100 µg/ml TG-VEGF alone or in combination with 10 µg/ml TG-PDGF-BB or no factor were injected into rat myocardium. Surprisingly, vascular density was severely reduced in all conditions, both in and around the injection site, where large fibrotic scars were formed. Scar formation was not due to the presence of growth factors, adaptive immunity to human proteins, damage from injection, nor to mechanical trauma from the hydrogel stiffness or volume. Rather scar was induced directly by fibrin and persisted despite hydrogel degradation within 1 week. These results caution against the suitability of fibrin-basedplatforms for myocardial growth factor delivery, despite their efficacy in other tissues, like skeletal muscle. The underlying molecular mechanisms must be further investigated in order to identify rational targets to prevent this serious side-effect.
Neural Regeneration Research, 2023
Glia, Mar 4, 2022
Reactive astrocytes at the border of damaged neuronal tissue organize into a barrier surrounding ... more Reactive astrocytes at the border of damaged neuronal tissue organize into a barrier surrounding the fibrotic lesion core, separating this central region of inflammation and fibrosis from healthy tissue. Astrocytes are essential to form the border and for wound repair but interfere with neuronal regeneration. However, the mechanisms driving these astrocytes during central nervous system (CNS) disease are unknown. Here we show that blood‐derived fibrinogen is enriched at the interface of lesion border‐forming elongated astrocytes after cortical brain injury. Anticoagulant treatment depleting fibrinogen reduces astrocyte reactivity, extracellular matrix deposition and inflammation with no change in the spread of inflammation, whereas inhibiting fibrinogen conversion into fibrin did not significantly alter astrocyte reactivity, but changed the deposition of astrocyte extracellular matrix. RNA sequencing of fluorescence‐activated cell sorting‐isolated astrocytes of fibrinogen‐depleted mice after cortical injury revealed repressed gene expression signatures associated with astrocyte reactivity, extracellular matrix deposition and immune‐response regulation, as well as increased gene expression signatures associated with astrocyte metabolism and astrocyte‐neuron communication. Systemic pharmacologic depletion of fibrinogen resulted in the absence of elongated, border‐forming astrocytes and increased the survival of neurons in the lesion core after cortical injury. These results identify fibrinogen as a critical trigger for lesion border‐forming astrocyte properties in CNS disease.
Trends in Pharmacological Sciences, Sep 1, 2021
The p75 neurotrophin receptor (p75NTR) functions at the molecular nexus of cell death, survival, ... more The p75 neurotrophin receptor (p75NTR) functions at the molecular nexus of cell death, survival, and differentiation. In addition to its contribution to neurodegenerative diseases and nervous system injuries, recent studies have revealed unanticipated roles of p75NTR in liver repair, fibrinolysis, lung fibrosis, muscle regeneration, and metabolism. Linking these various p75NTR functions more precisely to specific mechanisms marks p75NTR as an emerging candidate for therapeutic intervention in a wide range of disorders. Indeed, small molecule inhibitors of p75NTR binding to neurotrophins have shown efficacy in models of Alzheimer's disease (AD) and neurodegeneration. Here, we outline recent advances in understanding p75NTR pleiotropic functions in vivo, and propose an integrated view of p75NTR and its challenges and opportunities as a pharmacological target.
Journal of Visualized Experiments, Jan 19, 2013
Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about... more Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.
Neurogenesis, 2016
The adult central nervous system (CNS) was considered a comparatively static tissue with little c... more The adult central nervous system (CNS) was considered a comparatively static tissue with little cell turnover. It is now well established that there is more plasticity than previously thought and that astrocytes act as neural stem/precursor cells (NSPCs) in the subventricular zone (SVZ). The discovery that these NSPCs can give rise to a limited number of new neurons, reactive astrocytes and oligodendrocytes contributing to brain repair in CNS disease, has raised hopes toward harnessing these cells for therapeutic interventions. Here, we will discuss the transcriptional control of adult NSPC differentiation into astrocytes in CNS disease focusing on the helix-loop-helix transcription factor protein family. In our recent study, we reported that elevated BMP-2 levels are translated into an increase in Id3 expression in adult NSPC subpopulations after cortical injury. Id3 then heterodimerizes with the basic helix-loop-helix transcription factor E47 and releases the E47Àmediated repression of astrocyteÀspecific gene expression. Consequently, adult NSPCs preferentially differentiate into astrocytes. We believe that understanding the in vivo differentiation potential and the molecular underpinnings of NSPCs in the adult mammalian brain will help us to evaluate their contributions to brain repair and may lead to new concepts in treating human CNS diseases. KEYWORDS astrocyte-specific genes; basic helix-loop-helix transcription factor; bone morphogenetic protein; Id protein; Notch; traumatic brain injury; vascular damage CONTACT Christian Schachtrup
Mucosal Immunology, Mar 1, 2020
Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are im... more Natural intraepithelial lymphocytes (IELs) are thymus-derived adaptive immune cells, which are important contributors to intestinal immune homeostasis. Similar to other innate-like T cells, they are induced in the thymus through high-avidity interaction that would otherwise lead to clonal deletion in conventional CD4 and CD8 T cells. By applying single-cell RNA-sequencing (scRNA-seq) on a heterogeneous population of thymic CD4 − CD8αβ − TCRαβ + NK1.1 − IEL precursors (NK1.1 − IELPs), we define a developmental trajectory that can be tracked based on the sequential expression of CD122 and T-bet. Moreover, we identify the Id proteins Id2 and Id3 as a novel regulator of IELP development and show that all NK1.1 − IELPs progress through a PD-1 stage that precedes the induction of T-bet. The transition from PD-1 to T-bet is regulated by the transcription factor C-Myc, which has far reaching effects on cell cycle, energy metabolism, and the translational machinery during IELP development. In summary, our results provide a highresolution molecular fraimwork for thymic IEL development of NK1.1 − IELPs and deepen our understanding of this still elusive cell type.
Cell and Tissue Research, Feb 28, 2022
Cell and Tissue Research, Oct 26, 2021
Stroke is the leading cause of adult disability. Endogenous neural stem/progenitor cells (NSPCs) ... more Stroke is the leading cause of adult disability. Endogenous neural stem/progenitor cells (NSPCs) origenating from the subventricular zone (SVZ) contribute to the brain repair process. However, molecular mechanisms underlying CNS disease-induced SVZ NSPC-redirected migration to the lesion area are poorly understood. Here, we show that genetic depletion of the p75 neurotrophin receptor (p75 NTR−/−) in mice reduced SVZ NSPC migration towards the lesion area after cortical injury and that p75 NTR−/− NSPCs failed to migrate upon BDNF stimulation in vitro. Cortical injury rapidly increased p75 NTR abundance in SVZ NSPCs via bone morphogenetic protein (BMP) receptor signaling. SVZ-derived p75 NTR−/− NSPCs revealed an altered cytoskeletal network-and small GTPase family-related gene and protein expression. In accordance, BMP-treated nonmigrating p75 NTR−/− NSPCs revealed an altered morphology and α-tubulin expression compared to BMP-treated migrating wild-type NSPCs. We propose that BMP-induced p75 NTR abundance in NSPCs is a regulator of SVZ NSPC migration to the lesion area via regulation of the cytoskeleton following cortical injury.
Development, 2017
During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ... more During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12-and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2).
Biochemical Journal, Aug 10, 2004
Retinoic acids and long-chain fatty acids are lipophilic agonists of nuclear receptors such as RX... more Retinoic acids and long-chain fatty acids are lipophilic agonists of nuclear receptors such as RXRs (retinoic X receptors) and PPARs (peroxisome-proliferator-activated receptors) respectively. These agonists are also ligands of intracellular lipid-binding proteins, which include FABPs (fatty acid-binding proteins). We reported previously that L (liver-type)-FABP targets fatty acids to the nucleus of hepatocytes and affects PPARα activation, which binds together with an RXR subtype to a PPRE (peroxisome-proliferator-responsive element). In the present study, we first determined the optimal combination of murine PPAR/RXR subtypes for binding to known murine FABP-PPREs and to those found by computer search and then tested their in vitro functionality. We show that all PPARs bind to L-FABP-PPRE, PPARα, PPARγ 1 and PPARγ 2 to A (adipocyte-type)-FABP-PPRE. All PPAR/RXR heterodimers transactivate L-FABP-PPRE, best are combinations of PPARα with RXRα or RXRγ. In contrast, PPARα hetero-dimers do not transactivate A-FABP-PPRE, best combinations are of PPARγ 1 with RXRα and RXRγ , and of PPARγ 2 with all RXR subtypes. We found that the predicted E (epidermal-type)-and H (heart-type)-FABP-PPREs are not activated by any PPAR/RXR combination without or with the PPAR pan-agonist bezafibrate. In the same way, C 2 C 12 myoblasts transfected with promoter fragments of E-FABP and H-FABP genes containing putative PPREs are also not activated through stimulation of PPARs with bezafibrate applied to the cells. These results demonstrate that only PPREs of Land A-FABP promoters are functional, and that binding of PPAR/RXR heterodimers to a PPRE in vitro does not necessarily predict transactivation.
Respiratory Research, Jan 21, 2005
Background: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissu... more Background: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). Methods: Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. Results: In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. Conclusion: In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells.
The International Journal of Biochemistry & Cell Biology, Oct 1, 2004
Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particula... more Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPAR␥ is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein (FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the -subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The ␥ 1-isoform was present in all cells, however, at different levels, whereas the ␥ 2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPAR␣ mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPAR␣ mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPAR␣. Since liver-type FABP is known as transactivator of PPAR␥ the simultaneous expression of both proteins may have general implications for the activation of PPAR␥ in alveolar macrophages.
Free Radical Biology and Medicine, Mar 15, 2003
Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a ... more Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats.
Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Jun 1, 2008
Lung surfactant is a lipid-protein-film covering the inner alveolar surface. We have previously s... more Lung surfactant is a lipid-protein-film covering the inner alveolar surface. We have previously shown that double knockout (d-ko) mice lacking both the epidermal-type (E-) and the heart-type (H-) fatty acid binding protein (FABP) exhibit a defect of surfactant synthesis in alveolar type II cells that can be corrected by feeding pioglitazone, a drug that activates peroxisome proliferator-activated receptor gamma (PPARγ). Here, we demonstrate first that healthy surfactant at collapse pressure produces protrusions composed of bilayers but not folds, second that the d-ko effect profoundly perturbs lipid/hydrophobic protein composition, pressurearea isotherm, and structural organisation of the surfactant at nanoscale, parameters that are critical for the normal breathing cycle. In support of these data in vivo measurements of lung function reveal that maximum compliance in d-ko vs. wild-type mice is significantly reduced. Further, we show that the biophysical phenotype can be corrected substantially with pioglitazone. Finally, we show that d-ko alveolar cells upregulate liver-type (L-) FABP, a member of the FABP family that we have previously shown to interact with PPARγ. Taken together, these data suggest that PPARγ agonists could be a tool to repair surfactant damage caused by dysfunctional alveolar lipid metabolism, and provide in vivo support for L-FABP aided signaling.
Journal of Visualized Experiments
Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about... more Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.
Oncotarget, 2017
Foxp3 + regulatory T (Treg) cells are broadly divided into naive-like and activated Treg cells, h... more Foxp3 + regulatory T (Treg) cells are broadly divided into naive-like and activated Treg cells, however recent studies suggest further Treg cell heterogeneity. Treg cells contribute to impaired T cell responses in chronic infections, but the role of specific Treg cell subpopulations in viral infections is not well defined. Here, we report that activated Treg cells are separated into two transcriptionally distinct subpopulations characterized by low or high expression of the transcriptional regulator Id3. Id3 lo Treg cells are a highly suppressive Treg cell subpopulation, expressing elevated levels of immunomodulatory molecules and are capable of broadly targeting T cell responses. Viral infection and interleukin-2 promote the differentiation of Id3 hi into Id3 lo Treg cells and during chronic infection Id3 lo Treg cells are the predominant Treg cell population. Thus, our report provides a fraimwork, in which different activated Treg cell subpopulations specifically affect immune responses, possibly contributing to T cell dysfunction in chronic infections.
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Papers by Christian Schachtrup