Morten Meyer

Sensing of HSV-1 by the cGAS-STING pathway in microglia orchestrates antiviral defence in the CNS

Herpes simplex encephalitis (HSE) is the most common form of acute viral encephalitis in industrialized countries. Type I interferon (IFN) is important for control of herpes simplex virus (HSV-1) in the central nervous system (CNS). Here we show that microglia are the main source of HSV-induced type I IFN expression in CNS cells and these cytokines are induced in a cGAS–STING-dependent manner. Consistently, mice defective in cGAS or STING are highly susceptible to acute HSE. Although STING is redundant for cell-autonomous antiviral resistance in astrocytes and neurons, viral replication is strongly increased in neurons in STING-deficient mice. Interestingly, HSV-infected microglia confer STING-dependent antiviral activities in neurons and prime type I IFN production in astrocytes through the TLR3 pathway. Thus, sensing of HSV-1 infection in the CNS by microglia through the cGAS–STING pathway orchestrates an antiviral program that includes type I IFNs and immune-priming of other cell types.

Creation of a library of induced pluritent stem cell models from Parkinsonian patients

Induced pluripotent stem cells (iPSCs) are becoming an important source of pre-clinical models for research focusing on neurodegeneration. They offer the possibility for better understanding of common and divergent pathogenic mechanisms of brain diseases. Moreover, iPSCs provide a unique opportunity to develop personalized therapeutic strategies, as well as explore early pathogenic mechanisms, since they rely on the use of patients’ own cells that are otherwise accessible only post-mortem, when neuronal death-related cellular pathways and processes are advanced and adaptive. Neurodegenerative diseases are in majority of unknown cause, but mutations in specific genes can lead to familial forms of these diseases. For example, mutations in the superoxide dismutase 1 gene lead to the motor neuron disease amyotrophic lateral sclerosis (ALS), while mutations in the SNCA gene encoding for alpha-synuclein protein lead to familial Parkinson’s disease (PD). The generations of libraries of familial human ALS iPSC lines have been described, and the iPSCs rapidly became useful models for studying cell autonomous and non-cell autonomous mechanisms of the disease. Here we report the generation of a comprehensive library of iPSC lines of familial PD and an associated synucleinopathy, multiple system atrophy (MSA). In addition, we provide examples of relevant neural cell types these iPSC can be differentiated into, and which could be used to further explore early disease mechanisms. These human cellular models will be a valuable resource for identifying common and divergent mechanisms leading to neurodegeneration in PD and MSA.

Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia

Focal cerebral ischaemia has an initial phase of inflammation and tissue injury followed by a later phase of resolution and repair. Mass spectrometry imaging (desorption electrospray ionization and matrix assisted laser desorption ionization) was applied on brain sections from mice 2u2009h, 24u2009h, 5d, 7d, and 20d after permanent focal cerebral ischaemia. Within 24u2009h, N-acyl-phosphatidylethanolamines, lysophosphatidylcholine, and ceramide accumulated, while sphingomyelin disappeared. At the later resolution stages, bis(monoacylglycero)phosphate (BMP(22:6/22:6)), 2-arachidonoyl-glycerol, ceramide-phosphate, sphingosine-1-phosphate, lysophosphatidylserine, and cholesteryl ester appeared. At day 5 to 7, dihydroxy derivates of docosahexaenoic and docosapentaenoic acid, some of which may be pro-resolving mediators, e.g. resolvins, were found in the injured area, and BMP(22:6/22:6) co-localized with the macrophage biomarker CD11b, and probably with cholesteryl ester. Mass spectrometry imaging can visualize spatiotemporal changes in the lipidome during the progression and resolution of focal cerebral inflammation and suggests that BMP(22:6/22:6) and N-acyl-phosphatidylethanolamines can be used as biomarkers for phagocytizing macrophages/microglia cells and dead neurones, respectively.

Establishment and Characterization of a Tumor Stem Cell-Based Glioblastoma Invasion Model

Aims Glioblastoma is the most frequent and malignant brain tumor. Recurrence is inevitable and most likely connected to tumor invasion and presence of therapy resistant stem-like tumor cells. The aim was therefore to establish and characterize a three-dimensional in vivo-like in vitro model taking invasion and tumor stemness into account. Methods Glioblastoma stem cell-like containing spheroid (GSS) cultures derived from three different patients were established and characterized. The spheroids were implanted in vitro into rat brain slice cultures grown in stem cell medium and in vivo into brains of immuno-compromised mice. Invasion was followed in the slice cultures by confocal time-lapse microscopy. Using immunohistochemistry, we compared tumor cell invasion as well as expression of proliferation and stem cell markers between the models. Results We observed a pronounced invasion into brain slice cultures both by confocal time-lapse microscopy and immunohistochemistry. This invasion closely resembled the invasion in vivo. The Ki-67 proliferation indexes in spheroids implanted into brain slices were lower than in free-floating spheroids. The expression of stem cell markers varied between free-floating spheroids, spheroids implanted into brain slices and tumors in vivo. Conclusion The established invasion model kept in stem cell medium closely mimics tumor cell invasion into the brain in vivo preserving also to some extent the expression of stem cell markers. The model is feasible and robust and we suggest the model as an in vivo-like model with a great potential in glioma studies and drug discovery.

Convection-enhanced delivery of an anti-miR is well-tolerated, preserves anti-miR stability and causes efficient target de-repression: a proof of concept.

Over-expressed microRNAs (miRs) are promising new targets in glioblastoma (GBM) therapy. Inhibition of over-expressed miRs has been shown to diminish GBM proliferation, invasion and angiogenesis, indicating a significant therapeutic potential. However, the methods utilized for miR inhibition have had low translational potential. In clinical trials convection-enhanced delivery (CED) has been applied for local delivery of compounds in the brain. The aim of this study was to determine if safe and efficient miR inhibition was possible by CED of an anti-miR. We used a highly invasive GBM orthotopic xenograft model and targeted a well-validated miR, let-7a, with a 2′-O-methoxyethyl anti-miR with a combined phosphodiester/phosphorothioate backbone to establish an initial proof of concept. In vitro, anti-let-7a was delivered unassisted to the patient-derived T87 glioblastoma spheroid culture. In vivo, anti-let-7a or saline were administered by CED into orthotopic T87-derived tumors. After 1xa0month of infusion, tumors were removed and tumor mRNA levels of the target-gene High-mobility group AT-hook 2 (HMGA2) were determined. In vitro, 5xa0days inhibition was superior to 1xa0day at de-repressing the let-7a target HMGA2 and the inhibition was stable for 24xa0h. In vivo, anti-miR integrity was preserved in the pumps and no animals showed signs of severe adverse effects attributable to the anti-miR treatment. HMGA2 tumor level was significantly de-repressed in the anti-miR treated animals. The results showed—as an initial proof of concept—that miRs can be efficiently inhibited using CED delivery of anti-miR. The next step is to apply CED for anti-miR delivery focusing on key oncogenic miRs.

Influence of Oxygen Tension on Dopaminergic Differentiation of Human Fetal Stem Cells of Midbrain and Forebrain Origin

Neural stem cells (NSCs) constitute a promising source of cells for transplantation in Parkinsons disease (PD), but protocols for controlled dopaminergic differentiation are not yet available. Here we investigated the influence of oxygen on dopaminergic differentiation of human fetal NSCs derived from the midbrain and forebrain. Cells were differentiated for 10 days in vitro at low, physiological (3%) versus high, atmospheric (20%) oxygen tension. Low oxygen resulted in upregulation of vascular endothelial growth factor and increased the proportion of tyrosine hydroxylase-immunoreactive (TH-ir) cells in both types of cultures (midbrain: 9.1±0.5 and 17.1±0.4 (P<0.001); forebrain: 1.9±0.4 and 3.9±0.6 (P<0.01) percent of total cells). Regardless of oxygen levels, the content of TH-ir cells with mature neuronal morphologies was higher for midbrain as compared to forebrain cultures. Proliferative Ki67-ir cells were found in both types of cultures, but the relative proportion of these cells was significantly higher for forebrain NSCs cultured at low, as compared to high, oxygen tension. No such difference was detected for midbrain-derived cells. Western blot analysis revealed that low oxygen enhanced β-tubulin III and GFAP expression in both cultures. Up-regulation of β-tubulin III was most pronounced for midbrain cells, whereas GFAP expression was higher in forebrain as compared to midbrain cells. NSCs from both brain regions displayed less cell death when cultured at low oxygen tension. Following mictrotransplantation into mouse striatal slice cultures predifferentiated midbrain NSCs were found to proliferate and differentiate into substantial numbers of TH-ir neurons with mature neuronal morphologies, particularly at low oxygen. In contrast, predifferentiated forebrain NSCs microtransplanted using identical conditions displayed little proliferation and contained few TH-ir cells, all of which had an immature appearance. Our data may reflect differences in dopaminergic differentiation capacity and region-specific requirements of NSCs, with the dopamine-depleted striatum cultured at low oxygen offering an attractive micro-environment for midbrain NSCs.

Conditional ablation of myeloid TNF increases lesion volume after experimental stroke in mice, possibly via altered ERK1/2 signaling

Microglia are activated following cerebral ischemia and increase their production of the neuro- and immunomodulatory cytokine tumor necrosis factor (TNF). To address the function of TNF from this cellular source in focal cerebral ischemia we used TNF conditional knock out mice (LysMcreTNFfl/fl) in which the TNF gene was deleted in cells of the myeloid lineage, including microglia. The deletion reduced secreted TNF levels in lipopolysaccharide-stimulated cultured primary microglia by ~93%. Furthermore, phosphorylated-ERK/ERK ratios were significantly decreased in naïve LysMcreTNFfl/fl mice demonstrating altered ERK signal transduction. Micro-PET using 18[F]-fluorodeoxyglucose immediately after focal cerebral ischemia showed increased glucose uptake in LysMcreTNFfl/fl mice, representing significant metabolic changes, that translated into increased infarct volumes at 24u2009hours and 5 days compared to littermates (TNFfl/fl). In naïve LysMcreTNFfl/fl mice cytokine levels were low and comparable to littermates. At 6u2009hours, TNF producing microglia were reduced by 56% in the ischemic cortex in LysMcreTNFfl/fl mice compared to littermate mice, whereas no TNF+ leukocytes were detected. At 24u2009hours, pro-inflammatory cytokine (TNF, IL-1β, IL-6, IL-5 and CXCL1) levels were significantly lower in LysMcreTNFfl/fl mice, despite comparable infiltrating leukocyte populations. Our results identify microglial TNF as beneficial and neuroprotective in the acute phase and as a modulator of neuroinflammation at later time points after experimental ischemia, which may contribute to regenerative recovery.

Group i metabotropic glutamate receptors: A potential target for regulation of proliferation and differentiation of an immortalized human neural stem cell line

Human neural stem cells (NSCs) from the developing embryo or the subventricular zone of the adult brain can potentially elicit brain repair after injury or disease, either via endogenous cell proliferation or by cell transplantation. Profound knowledge of the diverse signals affecting these cells is, however, needed to realize their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and post‐natal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we differentiated an immortalized, forebrain‐derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists, MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I mGluRs reduced neurogenesis. Our results substantiate the importance of glutamate signalling in the regulation of human NSCs and may as such be applied to promote proliferation and neuronal differentiation.

Characterization of human neural differentiation from pluripotent stem cells using proteomics/PTMomics: current state-of-the-art and challenges

Stem cells are unspecialized cells capable of self‐renewal and to differentiate into the large variety of cells in the body. The possibility to differentiate these cells into neural precursors and neural cells in vitro provides the opportunity to study neural development, nerve cell biology, neurological disease as well as contributing to clinical research. The neural differentiation process is associated with changes at protein and their post‐translational modifications (PTMs). PTMs are important regulators of proteins physicochemical properties, function, activity, and interaction with other proteins, DNA/RNA, and complexes. Moreover, the interplay between PTMs is essential to regulate a range of cellular processes that abnormalities in PTM signaling are associated with several diseases. Altogether, this makes PTMs very relevant to study in order to uncover disease pathogenesis and increase the understanding of molecular processes in cells. Substantial advances in PTM enrichment methods and mass spectrometry has allowed the characterization of a subset of PTMs in large‐scale studies. This review focuses on the current state‐of‐the‐art of proteomic, as well as PTMomic studies related to human neural differentiation from pluripotent stem cells. Moreover, some of the challenges in stem cell biology, differentiation, and proteomics/PTMomics that are not exclusive to neural development will be discussed.

Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson’s Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson’s disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight of these lines (one of each SNCA, LRRK2 and GBA, four PARK2 lines, and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line, nor amongst the patient lines, although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast, iPSC, NSC, and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation, no mutation-specific clustering or difference was observed, though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis.