Friederike Schröter

Immunoproteasomes Preserve Protein Homeostasis upon Interferon-Induced Oxidative Stress

Interferon (IFN)-induced immunoproteasomes (i-proteasomes) have been associated with improved processing of major histocompatibility complex (MHC) class I antigens. Here, we show that i-proteasomes function to protect cell viability under conditions of IFN-induced oxidative stress. IFNs trigger the production of reactive oxygen species, which induce protein oxidation and the formation of nascent, oxidant-damaged proteins. We find that the ubiquitylation machinery is concomitantly upregulated in response to IFNs, functioning to target defective ribosomal products (DRiPs) for degradation by i-proteasomes. i-proteasome-deficiency in cells and in murine inflammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis. Efficient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our findings suggest that rather than having a specific role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis.

Sirt1 contributes critically to the redox-dependent fate of neural progenitors

Repair processes that are activated in response to neuronal injury, be it inflammatory, ischaemic, metabolic, traumatic or other cause, are characterized by a failure to replenish neurons and by astrogliosis. The underlying molecular pathways, however, are poorly understood. Here, we show that subtle alterations of the redox state, found in different brain pathologies, regulate the fate of mouse neural progenitor cells (NPCs) through the histone deacetylase (HDAC) Sirt1. Mild oxidation or direct activation of Sirt1 suppressed proliferation of NPCs and directed their differentiation towards the astroglial lineage at the expense of the neuronal lineage, whereas reducing conditions had the opposite effect. Under oxidative conditions in vitro and in vivo, Sirt1 was upregulated in NPCs, bound to the transcription factor Hes1 and subsequently inhibited pro-neuronal Mash1. In utero shRNA-mediated knockdown of Sirt1 in NPCs prevented oxidation-mediated suppression of neurogenesis and caused upregulation of Mash1 in vivo. Our results provide evidence for an as yet unknown metabolic master switch that determines the fate of neural progenitors.

Activation of kinin receptor B1 limits encephalitogenic T lymphocyte recruitment to the central nervous system

Previous proteomic and transcriptional analyses of multiple sclerosis lesions revealed modulation of the renin-angiotensin and the opposing kallikrein-kinin pathways. Here we identify kinin receptor B1 (Bdkrb1) as a specific modulator of immune cell entry into the central nervous system (CNS). We demonstrate that the Bdkrb1 agonist R838 (Sar-[D-Phe]des-Arg9-bradykinin) markedly decreases the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in SJL mice, whereas the Bdkrb1 antagonist R715 (Ac-Lys-[D-βNal7, Ile8]des-Arg9-bradykinin) resulted in earlier onset and greater severity of the disease. Bdkrb1-deficient (Bdkrb1−/−) C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein fragment, MOG35–55, showed more severe disease with enhanced CNS-immune cell infiltration. The same held true for mixed bone marrow–chimeric mice reconstituted with Bdkrb1−/− T lymphocytes, which showed enhanced T helper type 17 (TH17) cell invasion into the CNS. Pharmacological modulation of Bdkrb1 revealed that in vitro migration of human TH17 lymphocytes across blood-brain barrier endothelium is regulated by this receptor. Taken together, these results suggest that the kallikrein-kinin system is involved in the regulation of CNS inflammation, limiting encephalitogenic T lymphocyte infiltration into the CNS, and provide evidence that Bdkrb1 could be a new target for the treatment of chronic inflammatory diseases such as multiple sclerosis.

Dual roles of the adenosine A2a receptor in autoimmune neuroinflammation

BackgroundConditions of inflammatory tissue distress are associated with high extracellular levels of adenosine, due to increased adenosine triphosphate (ATP) degradation upon cellular stress or the release of extracellular ATP upon cell death, which can be degraded to adenosine by membrane-bound ecto-enzymes like CD39 and CD73. Adenosine is recognised to mediate anti-inflammatory effects via the adenosine A2a receptor (A2aR), as shown in experimental models of arthritis. Here, using pharmacological interventions and genetic inactivation, we investigated the roles of A2aR in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).MethodsWe used two independent mouse EAE variants, i.e. active immunization in C57BL/6 with myelin oligodendrocyte glycoprotein (MOG)35-55 or transfer-EAE by proteolipid protein (PLP)139-155-stimulated T lymphocytes and EAE in mice treated with A2aR-agonist CGS21680 at different stages of disease course and in mice lacking A2aR (A2aR−/−) compared to direct wild-type littermates. In EAE, we analysed myelin-specific proliferation and cytokine synthesis ex vivo, as well as inflammation and demyelination by immunohistochemistry. In vitro, we investigated the effect of A2aR on migration of CD4+ T cells, macrophages and microglia, as well as the impact of A2aR on phagocytosis of macrophages and microglia. Statistical tests were Mann-Whitney U and Student’s t test.ResultsWe found an upregulation of A2aR in the central nervous system (CNS) in EAE, predominantly detected on T cells and macrophages/microglia within the inflamed tissue. Preventive EAE treatment with A2aR-specific agonist inhibited myelin-specific T cell proliferation ex vivo and ameliorated disease, while application of the same agonist after disease onset exacerbated non-remitting EAE progression and resulted in more severe tissue destruction. Accordingly, A2aR-deficient mice showed accelerated and exacerbated disease manifestation with increased frequencies of IFN-γ-, IL-17- and GM-CSF-producing CD4+ T helper cells and higher numbers of inflammatory lesions in the early stage. However, EAE quickly ameliorated and myelin debris accumulation was lower in A2aR−/− mice. In vitro, activation of A2aR inhibited phagocytosis of myelin by macrophages and primary microglia as well as migration of CD4+ T cells, macrophages and primary microglia.ConclusionsA2aR activation exerts a complex pattern in chronic autoimmune neurodegeneration: while providing anti-inflammatory effects on T cells and thus protection at early stages, A2aR seems to play a detrimental role during later stages of disease and may thus contribute to sustained tissue damage within the inflamed CNS.

Immunoproteasomes Are Important for Proteostasis in Immune Responses

Document S1. Extended Experimental ProceduresxDownload (.11 MB ) Document S1. Extended Experimental Procedures

The proteasome complex and the maintenance of pluripotency: sustain the fate by mopping up?

The proteasome is a multi-enzyme complex responsible for orchestrating protein quality control by degrading misfolded, damaged, abnormal and foreign proteins. Studies related to the association of the proteasomal system in the preservation of self-renewal in both human and mouse pluripotent cells are sparse, and therefore a clear indication of the emergence of a new and important field of research. Under specific conditions the standard proteasome switches to the newly synthesized immunoproteasome, a catalytically active protein chamber also involved in the regulation of protein homeostasis, cell signaling and gene expression. Herein we review recent data to help elucidate and highlight the pivotal role of the proteasome complex, constitutive as well as inducible, in the regulation of self-renewal, pluripotency and differentiation of both embryonic and induced pluripotent stem cells. The proteasome that is endowed with enhanced proteolytic activity maintains self-renewal by regulating gene expression. In addition to protein degradation, the proteasome activator PA28, compartments of the 19S regulatory particle and key members of the ubiquitin pathway dictate the fate of a pluripotent stem cell. We anticipate that our observations will stimulate active research in this new and emerging theme related to stem cell biology, disease and regenerative medicine.

Lymphoblast-derived integration-free iPS cell line from a 69-year-old male

Human lymphoblast cells were used to generate integration-free induced pluripotent stem (iPS) cells employing episomal plasmids expressing OCT4, SOX2, NANOG, LIN28, C-MYC and L-MYC. The derived iPS cells were defined as pluripotent based on (i) expression of pluripotent-associated markers, (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptomes of the iPS cell line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95.

Activation of Wnt signaling promotes hippocampal neurogenesis in experimental autoimmune encephalomyelitis

BackgroundDisease progression in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), as one of its animal models, is characterized by demyelination and neuronal damage in white and gray matter structures, including the hippocampus. It is thought that dysfunction of the hippocampus, a primary locus of learning and memory consolidation, may contribute to cognitive impairment in MS patients. Previously, we reported an increased generation of hippocampal neuronal progenitors in the acute stage of EAE, whereas the microenvironmental signals triggering this process remained uninvestigated.ResultsIn the present study, we used the Wnt signaling reporter mouse Axin2LacZ, to elucidate the molecular mechanisms underlying the activation of the hippocampal neurogenic niche upon autoimmune neuroinflammation. Histological and enzymatic examinations of β-gal during the disease course of EAE, allowed us to survey hippocampal Wnt/β-catenin activity, one of the key signaling pathways of adult neurogenesis. We found that Wnt signaling is transiently upregulated in the acute stage of disease, consistent with a timely induction of canonical Wnt ligands. The enhancement of signaling coincided with hippocampal neuronal damage and local expression of immune cytokines such as TNFα and IFNγ, implicating the role of the inflammatory milieu in activation of the Wnt/β-catenin pathway. Supporting this finding, we show that transient exposure to pro-inflammatory cytokine TNFα triggers Wnt signaling in hippocampal organotypic slice cultures. Importantly, inflammation-mediated activation of the Wnt/β-catenin pathway was associated with enhanced neurogenesis in vitro and in vivo, indicating its potential role in hippocampal tissue regeneration and repair.ConclusionsThis study raises the possibility that enhancement of Wnt signaling may support neurogenic processes to cope with neuronal deficits upon immune-mediated neuroinflammation.

Lymphoblast-derived integration-free iPS cell line from a 65-year-old Alzheimer's disease patient expressing the TREM2 p.R47H variant

Human lymphoblast cells from a male patient diagnosed with Alzheimers disease (AD) expressing the TREM2 p.R47H variant were used to generate integration-free induced pluripotent stem (iPS) cells employing episomal plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The iPS cells retained the TREM2 mutation, and were defined as pluripotent based on (i) expression of pluripotent-associated markers, (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptomes of the iPS cell line and the human embryonic stem cell line H1 with a Pearson correlation of 0.966.

Lymphoblast-derived integration-free iPSC lines from a female and male Alzheimer's disease patient expressing different copy numbers of a coding CNV in the Alzheimer risk gene CR1

Human lymphoblast cells from a female and male patient diagnosed with Alzheimers disease (AD) with different genotypes of a functional copy number variation (CNV) in the AD risk gene CR1 were used to generate integration-free induced pluripotent stem cells (iPSCs) employing episomal plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The iPSCs retained the CR1 CNV, and comparative transcriptome analyses with the human embryonic stem cell line H1 revealed a Pearson correlation of 0.956 for AD1-CR10 and 0.908 for AD1-CR14.