Stephan Heermann

Microglia promote colonization of brain tissue by breast cancer cells in a Wnt-dependent way.

Although there is increasing evidence that blood‐derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf‐2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2‐like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor‐educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy.

Accumulation and clearance of α‐synuclein aggregates demonstrated by time‐lapse imaging

Aggregates of α‐synuclein are the pathological hallmark of sporadic Parkinson’s disease (PD), and mutations in the α‐synuclein gene underlie familial forms of the disease. To characterize the formation of α‐synuclein aggregates in living cells, we developed a new strategy to visualize α‐synuclein by fluorescence microscopy: α‐synuclein was tagged with a six amino acid PDZ binding motif and co‐expressed with the corresponding PDZ domain fused to enhanced green fluorescent protein (EGFP). In contrast to the traditional approach of α‐synuclein‐EGFP fusion proteins, this technique provided several‐fold higher sensitivity; this allowed us to compare α‐synuclein variants and perform time‐lapse imaging. A C‐terminally truncated α‐synuclein variant showed the highest prevalence of aggregates and toxicity, consistent with stabilization of the α‐synuclein monomer by its C‐terminus. Time‐lapse imaging illustrated how cells form and accumulate aggregates of α‐synuclein. A substantial number of cells also reduced their aggregate load, primarily through formation of an aggresome, which could itself be cleared from the cell. The molecular chaperone Hsp70 not only prevented the formation of aggregates, but also increased their reduction and clearance, underlining the therapeutic potential of similar strategies. In contrast to earlier assumptions build‐up, reduction and clearance of α‐synuclein aggregation thus appear a highly dynamic process.

Transforming Growth Factor β Cooperates with Persephin for Dopaminergic Phenotype Induction

The aim of the present study was to investigate the putative cooperative effects of transforming growth factor β (TGF‐β) and glial cell line‐derived neurotrophic factor (GDNF) family ligands in the differentiation of midbrain progenitors toward a dopaminergic phenotype. Therefore, a mouse midbrain embryonic day (E) 12 neurospheres culture was used as an experimental model. We show that neurturin and persephin (PSPN), but not GDNF, are capable of transient induction of dopaminergic neurons in vitro. This process, however, requires the presence of endogenous TGF‐β. In contrast, after 8 days in vitro GDNF rescued the TGF‐β neutralization‐dependent loss of the TH‐positive cells. In vivo, at E14.5, no apparent phenotype concerning dopaminergic neurons was observed in Tgf‐β2−/−/gdnf−/− double mutant mice. In vitro, combined TGF‐β/PSPN treatment achieved a yield of approximately 20% TH‐positive cells that were less vulnerable against 1‐methyl‐4‐phenyl pyridinium ion toxicity. The underlying TGF‐β/PSPN differentiation signaling is receptor‐mediated, involving p38 mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase pathways. These results indicate that phenotype induction and survival of fully differentiated neurons are accomplished through distinct pathways and individual factor requirement. TGF‐β is required for the induction of dopaminergic neurons, whereas GDNF is required for regulating and/or maintaining a differentiated neuronal phenotype. Moreover, this study suggests that the combination of TGF‐β with PSPN is a potent inductive cocktail for the generation of dopaminergic neurons that should be considered in tissue engineering and cell replacement therapies for Parkinsons disease.

TGF-β 1 enhances neurite outgrowth via regulation of proteasome function and EFABP

Malfunction of the ubiquitin-proteasome system has been implicated as a causal factor in the pathogenesis of aggregation-related disorders, e.g. Parkinsons disease. We show here that Transforming growth factor-beta 1 (TGF-beta), a multifunctional cytokine and trophic factor for dopaminergic (DAergic) neurons modulates proteasome function in primary midbrain neurons. TGF-beta differentially inhibited proteasomal subactivities with a most pronounced time-dependent inhibition of the peptidyl-glutamyl peptide hydrolyzing-like and chymotrypsin-like subactivity. Regulation of proteasomal activity could be specifically quantified in the DAergic subpopulation. Protein blot analysis revealed an accumulation of ubiquitinated proteins after TGF-beta treatment. The identity of these enriched proteins was further analyzed by 2D-gel electrophoresis and mass spectrometry. We found epidermal fatty acid binding protein (EFABP) to be strongly increased and ubiquitinated after TGF-beta treatment and confirmed this finding by co-immunoprecipitation. While application of TGF-beta increased neurite regeneration in a scratch lesion model, downregulation of EFABP by siRNA significantly decreased this effect. We thus postulate that a differential regulation of proteasomal function, as demonstrated for TGF-beta, can result in an enrichment of proteins, such as EFABP, that mediate physiological functions, such as neurite regeneration.

Eye morphogenesis driven by epithelial flow into the optic cup facilitated by modulation of bone morphogenetic protein

The hemispheric, bi-layered optic cup forms from an oval optic vesicle during early vertebrate eye development through major morphological transformations. The overall basal surface, facing the developing lens, is increasing, while, at the same time, the space basally occupied by individual cells is decreasing. This cannot be explained by the classical view of eye development. Using zebrafish (Danio rerio) as a model, we show that the lens-averted epithelium functions as a reservoir that contributes to the growing neuroretina through epithelial flow around the distal rims of the optic cup. We propose that this flow couples morphogenesis and retinal determination. Our 4D data indicate that future stem cells flow from their origin in the lens-averted domain of the optic vesicle to their destination in the ciliary marginal zone. BMP-mediated inhibition of the flow results in ectopic neuroretina in the RPE domain. Ultimately the ventral fissure fails to close resulting in coloboma. DOI: http://dx.doi.org/10.7554/eLife.05216.001

Organization of glomeruli in the main olfactory bulb of Xenopus laevis tadpoles

Structural and functional investigations were carried out to study olfactory glomeruli in the main olfactory bulb (OB) in tadpoles of the clawed frog, Xenopus laevis. Calcium imaging of odor response patterns of OB neurons revealed that the synapses within the glomeruli are functional. Tracing axons of individual olfactory receptor neurons (ORNs), dendrites of mitral/tufted (M/T) cells and processes of periglomerular interneurons indicate that the glomerular architecture is solely determined by terminal branches of ORN axons and tufts of M/T primary dendrites. The small population of periglomerular neurons forms wide‐field arborizations that always extend over many glomeruli, enter the glomeruli, but lack any glomerular tufts. Antibodies to synaptophysin indicate a high density of synapses within glomeruli, which was further confirmed at the ultrastructural level and quantified to approximately 0.5 synaptic sites per μm2. Combining immunocytochemistry and ultrastructural investigations, we show that glomeruli in Xenopus laevis tadpoles lack any cellular borders. Glomeruli are surrounded neither by periglomerular somata nor by glial processes. Taken together, our results demonstrate that olfactory glomeruli in Xenopus laevis tadpoles (1) are fully functional, (2) are spheroidal neuropil aggregations of terminal tufts of ORNs and tufts of primary dendrites of M/T cells, and (3) are not enwrapped by a border formed by juxtaglomerular cells. J. Comp. Neurol. 464:257–268, 2003.

Molecular control of Schwann cell migration along peripheral axons

The development of the peripheral nervous system (PNS) is a highly dynamic process, during which motor and sensory axons innervate distal targets, such as skeletal muscles and skin. Axonal function depends critically on support from Schwann cells, the main glial cell type in the PNS. Schwann cells originate from the neural crest, migrate along outgrowing axons and associate with axons along their entire length prior to ensheathment or myelination. How axonal growth and the migration of Schwann cells is coordinated at the level of reciprocal axon-glial signaling is the fascinating subject of ongoing research. Neuregulin-1 (NRG1) type III, an axonal membrane-bound ligand for receptor tyrosine kinases of the ErbB family, acts as a “master regulator” of peripheral myelination. In addition, NRG1-ErbB signaling directs the development of the Schwann cell lineage and regulates the proliferation and survival of Schwann cells. Studies in zebrafish have identified a direct role of NRG1 type III in Schwann cell migration, but to what extend NRG1 serves a similar function in the mammalian PNS is not clear. We have employed a mouse superior cervical ganglion explant culture system, in which the migration of endogenous Schwann cells along outgrowing axons can be visualized by time-lapse imaging. Using this approach, we found that NRG1 type III-ErbB signaling regulates the colonization of distal axonal segments by Schwann cells. However, our data suggest an indirect effect of NRG1 type III-ErbB signaling via the support of Schwann cell survival in proximal axonal regions rather than a direct effect on Schwann cell motility.

Presynaptic protein distribution and odour mapping in glomeruli of the olfactory bulb of Xenopus laevis tadpoles

The sensory input layer in the olfactory bulb (OB) is typically organized into spheroidal aggregates of dense neuropil called glomeruli. This characteristic compartmentalization of the synaptic neuropil is a typical feature of primary olfactory centres in vertebrates and most advanced invertebrates. In the present work we mapped the location of presynaptic sites in glomeruli across the OB using antibodies to presynaptic vesicle proteins and presynaptic membrane proteins in combination with confocal microscopy. In addition the responses of glomeruli upon mucosal application of amino acid‐odorants and forskolin were monitored using functional calcium imaging. We first describe the spatial distribution of glomeruli across the main olfactory bulb (MOB) in premetamorphic Xenopus laevis. Second, we show that the heterogeneous organization of glomeruli along the dorsoventral and mediolateral axes of the MOB is associated with a differential distribution of synaptic vesicle proteins. While antibodies to synaptophysin, syntaxin and SNAP‐25 uniformly labelled glomeruli in the whole MOB, intense synaptotagmin staining was present only in glomeruli in the lateral, and to a lesser extent in the intermediate, part of the OB. Interestingly, amino acid‐responsive glomeruli were always located in the lateral part of the OB, and glomeruli activated by mucosal forskolin application were exclusively located in the medial part of the OB. This correlation between odour mapping and presynaptic protein distribution is an additional hint on the existence of different subsystems within the main olfactory system in larval Xenopus laevis.

In vivo requirement of TGF-β/GDNF cooperativity in mouse development : focus on the neurotrophic hypothesis

Neurotrophic factors are well‐recognized extracellular signaling molecules that regulate neuron development including neurite growth, survival and maturation of neuronal phenotypes in the central and peripheral nervous system. Previous studies have suggested that TGF‐β plays a key role in the regulation of neuron survival and death and potentiates the neurotrophic activity of several neurotrophic factors, most strikingly of GDNF. To test the physiological relevance of this finding, TGF‐β2/GDNF double mutant (d‐ko) mice were generated. Double mutant mice die at birth like single mutants due to kidney agenesis (GDNF−/−) and congential cyanosis (TGF‐β2−/−), respectively. To test for the in vivo relevance of TGF‐β2/GDNF cooperativity to regulate neuron survival, mesencephalic dopaminergic neurons, lumbar motoneurons, as well as neurons of the lumbar dorsal root ganglion and the superior cervical ganglion were investigated. No loss of mesencephalic dopaminergic neurons was observed in double mutant mice at E18.5. A partial reduction in neuron numbers was observed in lumbar motoneurons, sensory and sympathetic neurons in GDNF single mutants, which was further reduced in TGF‐β2/GDNF double mutant mice at E18.5. However, TGF‐β2 single mutant mice showed no loss of neurons. These data point towards a cooperative role of TGF‐β2 and GDNF with regard to promotion of survival within the peripheral motor and sensory systems investigated.

Neuregulin 1 Type III/ErbB Signaling Is Crucial for Schwann Cell Colonization of Sympathetic Axons

Analysis of Schwann cell (SC) development has been hampered by the lack of growing axons in many commonly used in vitro assays. As a consequence, the molecular signals and cellular dynamics of SC development along peripheral axons are still only poorly understood. Here we use a superior cervical ganglion (SCG) explant assay, in which axons elongate after treatment with nerve growth factor (NGF). Migration as well as proliferation and apoptosis of endogenous SCG-derived SCs along sympathetic axons were studied in these cultures using pharmacological interference and time-lapse imaging. Inhibition of ErbB receptor tyrosine kinases leads to reduced SC proliferation, increased apoptosis and thereby severely interfered with SC migration to distal axonal sections and colonization of axons. Furthermore we demonstrate that SC colonization of axons is also strongly impaired in a specific null mutant of an ErbB receptor ligand, Neuregulin 1 (NRG1) type III. Taken together, using a novel SC development assay, we demonstrate that NRG1 type III serves as a critical axonal signal for glial ErbB receptors that drives SC development along sympathetic axons.