Stefan Schumacher

Regulation of miRNA expression during neural cell specification

MicroRNA (miRNA) are a newly recognized class of small, noncoding RNA molecules that participate in the developmental control of gene expression. We have studied the regulation of a set of highly expressed neural miRNA during mouse brain development. Temporal control is a characteristic of miRNA regulation in C. elegans and Drosophila, and is also prominent in the embryonic brain. We observed significant differences in the onset and magnitude of induction for individual miRNAs. Comparing expression in cultures of embryonic neurons and astrocytes we found marked lineage specificity for each of the miRNA in our study. Two of the most highly expressed miRNA in adult brain were preferentially expressed in neurons (mir‐124, mir‐128). In contrast, mir‐23, a miRNA previously implicated in neural specification, was restricted to astrocytes. mir‐26 and mir‐29 were more strongly expressed in astrocytes than neurons, others were more evenly distributed (mir‐9, mir‐125). Lineage specificity was further explored using reporter constructs for two miRNA of particular interest (mir‐125 and mir‐128). miRNA‐mediated suppression of both reporters was observed after transfection of the reporters into neurons but not astrocytes. miRNA were strongly induced during neural differentiation of embryonic stem cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development.

Post-transcriptional regulation of the let-7 microRNA during neural cell specification

The let‐7 miRNA regulates developmental timing in C. elegans and is an important paradigm for investigations of miRNA functions in mammalian development. We have examined the role of miRNA precursor processing in the temporal control and lineage specificity of the let‐7 miRNA. In situ hybridization (ISH) in E9.5 mouse embryos revealed early induction of let‐7 in the developing central nervous system. The expression pattern of three let‐7 family members closely resembled that of the brain‐enriched miRNAs mir‐124, mir‐125 and mir‐128. Comparison of primary, precursor, and mature let‐7 RNA levels during both embryonic brain development and neural differentiation of embryonic stem cells and embryocarcinoma (EC) cells suggest post‐transcriptional regulation of let‐7 accumulation. Reflecting these results, let‐7 sensor constructs were strongly down‐regulated during neural differentiation of EC cells and displayed lineage specificity in primary cells. Neural differentiation of EC cells was accompanied by an increase in let‐7 precursor processing activity in vitro. Furthermore, undifferentiated and differentiated cells contained distinct precursor RNA binding complexes. A neuron‐enhanced binding complex was shown by antibody challenge to contain the miRNA pathway proteins Argonaute1 and FMRP. Developmental regulation of the processing pathway correlates with differential localization of the proteins Argonaute, FMRP, MOV10, and TNRC6B in self‐renewing stem cells and neurons.—Wulczyn, F. G., Smirnova, L., Rybak, A., Brandt, C., Kwidzinski, E., Ninnemann, O., Strehle, M., Seiler, A., Schumacher, S., Nitsch, R. Post‐transcriptional regulation of the let‐7 microRNA during neural cell specification. FASEB J. 21, 415–426 (2007)

miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR‐124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS‐specific microRNA miR‐124 and connect the regulation of RhoG expression by miR‐124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR‐124 to control axonal and dendritic branching.

Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development.

UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4s subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the proteins possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.

The neural EGF family member CALEB/NGC mediates dendritic tree and spine complexity.

The development of dendritic arborizations and spines is essential for neuronal information processing, and abnormal dendritic structures and/or alterations in spine morphology are consistent features of neurons in patients with mental retardation. We identify the neural EGF family member CALEB/NGC as a critical mediator of dendritic tree complexity and spine formation. Overexpression of CALEB/NGC enhances dendritic branching and increases the complexity of dendritic spines and filopodia. Genetic and functional inactivation of CALEB/NGC impairs dendritic arborization and spine formation. Genetic manipulations of individual neurons in an otherwise unaffected microenvironment in the intact mouse cortex by in utero electroporation confirm these results. The EGF‐like domain of CALEB/NGC drives both dendritic branching and spine morphogenesis. The phosphatidylinositide 3‐kinase (PI3K)‐Akt‐mammalian target of rapamycin (mTOR) signaling pathway and protein kinase C (PKC) are important for CALEB/NGC‐induced stimulation of dendritic branching. In contrast, CALEB/NGC‐induced spine morphogenesis is independent of PI3K but depends on PKC. Thus, our findings reveal a novel switch of specificity in signaling leading to neuronal process differentiation in consecutive developmental events.

CALEB/NGC Interacts with the Golgi-associated Protein PIST

CALEB/NGC is a neural member of the epidermal growth factor protein family expressed in axon and synapse-rich areas of the nervous system and shown to be important for neurite formation. It can bind to the extracellular matrix proteins tenascin-R and tenascin-C. Here we show that CALEB/NGC interacts with the Golgi-associated protein PIST. PIST was originally described as an interaction partner of the small GTPase TC10 and was then found to be Golgi-associated by binding to syntaxin-6 and to be important for the transport of frizzled proteins and the cystic fibrosis transmembrane conductance regulator to the plasma membrane. In addition, PIST was demonstrated to be involved in autophagy and linked to processes of neurodegeneration. CALEB/NGC interacts with PIST in the yeast two-hybrid system. This interaction can be confirmed by co-immunoprecipitations and co-localization studies. The juxtamembrane cytoplasmic peptide segment of CALEB/NGC, highly conserved during evolution, mediates the binding to PIST. CALEB/NGC co-localizes with PIST in the Golgi apparatus of transfected COS7 cells and in Golgi-derived vesicles after brefeldin A or nocodazole treatment. Co-localization studies in primary hippocampal cells and analysis of Purkinje cells of colchicine-treated rats, serving as an in vivo model system to block microtubule-dependent transport processes, support the view that PIST is an interaction partner of CALEB/NGC and implicate that this interaction may play a role in the intracellular transport of CALEB/NGC.

Regulated binding of the fibrinogen-like domains of tenascin-R and tenascin-C to the neural EGF family member CALEB

The neural transmembrane protein CALEB was discovered in a screen for novel molecules implicated in neuronal differentiation processes and was found to bind to two proteins of the extracellular matrix, tenascin‐C and tenascin‐R. The expression of different isoforms of CALEB in axon‐ and synapse‐rich areas in the nervous system is regulated during development. Here we show that an unusual acidic peptide segment of CALEB is sufficient to mediate the binding of CALEB to the fibrinogen‐like globes of both tenascin family members as well as to native tenascin‐C. We idengify a small sequence element within the acidic peptide segment of CALEB as important for this binding. Interestingly, the interactions of CALEB and tenascin‐C and ‐R seem to be regulated during development. We demonstrate that only CALEB‐80, the expression of which is up‐regulated in the chicken retina during synaptogenesis, but not CALEB‐140, expressed later on in development, can bind to the fibrinogen‐like domains of tenascin‐R or tenascin‐C and to native tenascin‐C. While both CALEB‐80 and CALEB‐140 are expressed in the plexiform layers and the optic fiber layer of embryonic chicken retina, CALEB‐140 labeling is more intense in the optic fiber layer in comparison to the inner plexiform layer.

miR-124-regulated RhoG: A conductor of neuronal process complexity

RhoG is a member of the Rho family of small GTPases sharing highest sequence similarity with Rac and Cdc42. Mig-2 and Mtl represent the functional equivalents of RhoG in Caenorhabditis elegans and Drosophila, respectively. RhoG has attracted great interest because it plays a central role in the regulation of cytoskeletal reorganization in various physiological and pathophysiological situations. For example, it is fundamental to phagocytotic processes, is able to regulate gene expression, cell survival and proliferation, and is involved in cell migration and in the invasion of pathogenic bacteria. The activation of Rac1 via an ELMO/Dock180 module has been elaborated to be important for RhoG signaling. Although a stimulatory role for neurite outgrowth in the pheochromocytoma PC12 cell line has been assigned to RhoG, the exact function of this GTPase for the development of the processes of primary neurons remains to be clarified. In this view, we discuss the impact of RhoG on axonal and dendritic differentiation, its role as a conductor of Rac1 and Cdc42 activity and the functional regulation of RhoG expression by the microRNA miR-124.

B56β, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC-mediated dendritic branching

The development of dendritic arbors is critical in neuronal circuit formation, as dendrites are the primary sites of synaptic input. Morphologically specialized dendritic protrusions called spines represent the main postsynaptic compartment for excitatory neurotransmission. Recently, we demonstrated that chicken acidic leucine‐rich epidermal growth factor (EGF) ‐like domain‐containing brain protein/neuroglycan C (CALEB/ NGC), a neural member of the EGF family, mediates dendritic tree and spine complexity but that the signaling pathways in the respective processes differ. For a more detailed characterization of these signal transduction pathways, we performed a yeast two‐hybrid screen to identify proteins that interact with CALEB/NGC. Our results show that B56β, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC‐mediated dendritic branching but not spine formation. Binding of B56β to CALEB/NGC was confirmed by several biochemical and immunocytochemical assays. Using affinity chromatography and mass spectrometry, we demonstrate that the whole protein phosphatase 2A trimer, including structural and catalytic subunits, binds to CALEB/NGC via B56β. We show that CALEB/ NGC induces the phosphorylation of Akt in dendrites. Previously described to interfere with Akt signaling, B56β inhibits Akt phosphorylation and Akt‐dependent dendritic branching but not Akt‐independent spine formation induced by CALEB/NGC. Our results contribute to a better understanding of signaling specificity leading to neuronal process differentiation in sequential developmental events.—Brandt, N., Franke, K., Johannes, S., Buck, F., Harder, S., Hassel, B., Nitsch, R., Schumacher, S. B56β, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC‐mediated dendritic branching. FASEB J. 22, 2521–2533 (2008)

Different roles of the small GTPases Rac1, Cdc42, and RhoG in CALEB/NGC-induced dendritic tree complexity

Rho GTPases play prominent roles in the regulation of cytoskeletal reorganization. Many aspects have been elaborated concerning the individual functions of Rho GTPases in distinct signaling pathways leading to cytoskeletal rearrangements. However, major questions have yet to be answered regarding the integration and the signaling hierarchy of different Rho GTPases in regulating the cytoskeleton in fundamental physiological events like neuronal process differentiation. Here, we investigate the roles of the small GTPases Rac1, Cdc42, and RhoG in defining dendritic tree complexity stimulated by the transmembrane epidermal growth factor family member CALEB/NGC. Combining gain‐of‐function and loss‐of‐function analysis in primary hippocampal neurons, we find that Rac1 is essential for CALEB/NGC‐mediated dendritic branching. Cdc42 reduces the complexity of dendritic trees. Interestingly, we identify the palmitoylated isoform of Cdc42 to adversely affect dendritic outgrowth and dendritic branching, whereas the prenylated Cdc42 isoform does not. In contrast to Rac1, CALEB/NGC and Cdc42 are not directly interconnected in regulating dendritic tree complexity. Unlike Rac1, the Rac1‐related GTPase RhoG reduces the complexity of dendritic trees by acting upstream of CALEB/NGC. Mechanistically, CALEB/NGC activates Rac1, and RhoG reduces the amount of CALEB/NGC that is located at the right site for Rac1 activation at the cell membrane. Thus, Rac1, Cdc42, and RhoG perform very specific and non‐redundant functions at different levels of hierarchy in regulating dendritic tree complexity induced by CALEB/NGC.