Robert Glumm

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.

Lower motor neuron loss in multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is considered a chronic inflammatory and demyelinating disease of the central nervous system. Evidence that axonal and neuronal pathology contributes to the disease is accumulating, however, the distribution of neuronal injury as well as the underlying mechanisms have not yet been fully clarified. Here, we investigated the role of neuronal cell loss in MS and its animal model, experimental autoimmune encephalomyelitis (EAE).

CNS-irrelevant T-cells enter the brain, cause blood–brain barrier disruption but no glial pathology

Invasion of autoreactive T‐cells and alterations of the blood–brain barrier (BBB) represent early pathological manifestations of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Non‐CNS‐specific T‐cells are also capable of entering the CNS. However, studies investigating the spatial pattern of BBB alterations as well as the exact localization and neuropathological consequences of transferred non‐CNS‐specific cells have been thus far lacking. Here, we used magnetic resonance imaging and multiphoton microscopy, as well as histochemical and high‐precision unbiased stereological analyses to compare T‐cell transmigration, localization, persistence, relation to BBB disruption and subsequent effects on CNS tissue in a model of T‐cell transfer of ovalbumin (OVA)‐ and proteolipid protein (PLP)‐specific T‐cells. BBB alterations were present in both EAE‐mice and mice transferred with OVA‐specific T‐cells. In the latter case, BBB alterations were less pronounced, but the pattern of initial cell migration into the CNS was similar for both PLP‐ and OVA‐specific cells [mean (SEM), 95 × 103 (7.6 × 103) and 88 × 103 (18 × 103), respectively]. Increased microglial cell density, astrogliosis and demyelination were, however, observed exclusively in the brain of EAE‐mice. While mice transferred with non‐neural‐specific cells showed similar levels of rhodamine‐dextran extravasation in susceptible brain regions, EAE‐mice presented huge BBB disruption in brainstem and moderate leakage in cerebellum. This suggests that antigen specificity and not the absolute number of infiltrating cells determine the magnitude of BBB disruption and glial pathology.

In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

BackgroundTwo-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.MethodsWe applied ex vivo and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.ResultsBy applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.ConclusionsThe activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.

Characterization of natural killer cells in paired CSF and blood samples during neuroinflammation

Natural killer (NK) cells from paired CSF and blood samples of patients with multiple sclerosis (MS), other neuroinflammatory diseases (IND), and non-inflammatory neurological diseases (NIND) were characterized using flow cytometry. NK cell frequency in CSF was overall decreased compared to blood, particularly in MS patients. In contrast to blood NK cells, during neuroinflammation, CSF NK cells display an immature phenotype with bright expression of CD56 and CD27 and reduced CX3CR1 expression. Our findings suggest that, as for central memory T cells, CSF may represent an intermediary compartment for NK cell trafficking and differentiation before entering the CNS parenchyma.

Homeostatic regulation of NCAM polysialylation is critical for correct synaptic targeting

During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation. Using a chemically modified sialic acid precursor (N-propyl-d-mannosamine), we inhibited the polysialyltransferase ST8SiaII, the principal enzyme involved in polysialylation during development, at selected developmental time-points. This treatment altered NCAM polysialylation while NCAM expression was not affected. Altered polysialylation resulted in an aberrant mossy fiber projection that formed glutamatergic terminals on pyramidal neurons of the CA1 region in organotypic slice cultures and in vivo. Electrophysiological recordings revealed that the ectopic terminals on CA1 pyramids were functional and displayed characteristics of mossy fiber synapses. Moreover, ultrastructural examination indicated a “mossy fiber synapse”-like morphology. We thus conclude that homeostatic regulation of the amount of synthesized polysialic acid at specific developmental stages is essential for correct synaptic targeting and circuit formation during hippocampal development.

Impaired postnatal development of hippocampal neurons and axon projections in the Emx2-/- mutants.

The specification and innervation of cerebral subregions is a complex layer‐specific process, primed by region‐specific transcription factor expression and axonal guidance cues. In Emx2–/– mice, the hippocampus fails to form a normal dentate gyrus as well as the normal layering of principal neurons in the hippocampus proper. Here, we analyzed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking Emx2 expression in vitro. As these mutants die perinatally, we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In late embryonic Emx2–/– cultivated hippocampi, both the perforant path as well as the distribution of calretinin‐positive cells are affected. Traced entorhinal afferents in co‐cultures with hippocampus from embryonic Emx2–/– mice terminate diffusely in the prospective dentate gyrus in contrast to the layer‐specific termination of co‐cultures from wild‐type littermates. In addition, in brain slice cultures from null mutants the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. Our data indicate that Emx2 is essential for the terminal differentiation of granular cells and the correct formation of extrinsic and intrinsic hippocampal connections.

Second Symposium on Normal and Abnormal Development of the Human Fetal Brain

Neuroembryology 2002;1:191–193 DOI: 10.1159/000066269 Second Symposium on Normal and Abnormal Development of the Human Fetal Brain The University of Rostock, Neuroembryonic Research Laboratory, Department of Anatomy, Rostock, Germany