Hansjürgen Volkmer

Organization of the Neurofascin Gene and Analysis of Developmentally Regulated Alternative Splicing

Neurofascin is an axonal member of the L1 subgroup of the immunoglobulin superfamily implicated in neurite extension in the course of embryonic development. Here we have isolated and characterized the gene encoding chicken neurofascin. Comparison of genomic sequences with cDNA sequences provides the structure and localization of intron/exon boundaries and indicates that neurofascin isoforms are generated by alternative splicing of its pre-mRNA. The neurofascin gene is composed of 33 exons distributed over 72 kilobases. Each of the six immunoglobulin- and five fibronectin-type III-like domains is encoded by two exons. While introns between domains are of phase 1, others are of phase 0, 1, or 2. Alternative splicing of neurofascin is developmentally regulated as shown by polymerase chain reaction analysis. Furthermore, plasmid libraries from long range polymerase chain reaction-amplified cDNA of neurofascin were used to examine and quantify the distribution of alternatively spliced exons in individual neurofascin molecules. We found 50 different neurofascin isoforms at different developmental stages and revealed the existence of one major “early” in comparison with multiple “late” neurofascin isoforms.

Neurofascin regulates the formation of gephyrin clusters and their subsequent translocation to the axon hillock of hippocampal neurons

Little is known about the role of cell adhesion molecules (CAMs) in inhibitory synapse development. In particular, a functional link between CAMs and the clustering of postsynaptic scaffold component gephyrin, which is a critical determinant of gamma-aminobutyric acid A (GABA) receptor clustering, still needs to be elaborated. At early stages of inhibitory synapse formation, gephyrin and CAM neurofascin are diffusely expressed in the soma of hippocampal neurons. Subsequently, gephyrin clusters become localized to the axon hillock and neurofascin is observed all over the soma including the axon hillock suggesting a function for neurofascin in gephyrin clustering. Transfection of expression vectors for different isoforms and mutants of neurofascin revealed that neurofascin is required for the formation of gephyrin clusters presumably dependent on extracellular interactions. Furthermore, expression of neurofascin is necessary for the translocation of gephyrin clusters to the axon hillock of hippocampal neurons as shown by shRNA-mediated knockdown. In addition, overexpression of an embryonic neurofascin isoform is sufficient for functional rescue after knockdown of endogenous neurofascin.

Neurofascin: a switch between neuronal plasticity and stability.

Neurofascin (NF) is a cell surface protein belonging to the immunoglobulin superfamily (IgSF). Different polypeptides of 186, 180, 166 and 155 kDa are generated by alternative splicing. Expression of these isoforms is temporally and spatially regulated and can be roughly grouped into embryonic, adult and glial expression. NF interacts with many different interaction partners both extra- and intracellularly. Interactions of NF166 and NF180 selectively regulate mechanisms of plasticity like neurite outgrowth and the formation postsynaptic components. By contrast, NF155 and NF186 confer stabilization of neural structures by interaction with voltage-gated sodium channels and ankyrinG at axon initial segments (AIS) or nodes of Ranvier as well as neuron-glia interactions at the paranodes. Alternatively spliced isoforms of neurofascin may therefore balance dynamic and stabilizing mechanisms of the CNS.

Stress modulation of hippocampal activity – Spotlight on the dentate gyrus

The effects of stress on learning and memory are diverse, ranging from impairment to facilitation. Many studies emphasize the major role of the hippocampus, mainly its CA1 and CA3 areas, in the process of memory formation under emotional and stressful conditions. In the current review, we summarize work which suggests that the dentate gyrus (DG) of the hippocampus is likely to play a pivotal role in defining the impact of stress on hippocampal functioning. We describethe effects of stress on long term potentiation (LTP) and local circuit activity in the DG and the role of the amygdala in mediating these effects. As one of the brain regions known to have a high rate of adult neurogenesis, the effects of stress on DG neurogenesis will also be reviewed. Finally, we discuss exposure to stress during juvenility and its influence on the adult DG. The DG is a dynamic structure which is susceptible to stress. Under stressful conditions, its response is variable and complex, much like the behavioral outcomes of such circumstances. It is likely to significantly contribute to the diverse effects of stress on memory formation.

A Comprehensive Small Interfering RNA Screen Identifies Signaling Pathways Required for Gephyrin Clustering

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABAA receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)–Akt also activates mTOR and represses GSK3β, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF–mTOR signaling is associated with reduced GABAA receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K–Akt–mTOR signaling.

A regulated switch of chick neurofascin isoforms modulates ligand recognition and neurite extension

Neural cell adhesion molecule neurofascin regulates the induction of neurite outgrowth, the establishment of synaptic connectivity and myelination. Neurofascin isoforms are generated by spatially and temporally controlled alternative splicing. Isoform NF166 is predominantly expressed in dorsal root ganglia from embryonal day 5 (E5) to E8, and a further neurofascin isoform NF185 appears at E9. Expression of neurofascin and its binding partner axonin-1 on sensory fibers implies functional interactions for neurite outgrowth. E7 sensory neurons require NF166-axonin-1 interactions for neurite extension, accordingly. The contribution of NF166-axonin-1 interaction for neurite outgrowth decreases in parallel with the appearance of NF185 on sensory neurons at E9. This finding may be explained by (1) alleviated intrinsic capability to use axonin-1 as a cellular receptor and (2) reduced binding of axonin-1 to NF185. Finally, NF166, but not NF185, serves as a cellular receptor for neurite induction via homophilic interactions with a neurofascin substrate.

Homophilic interactions of chick neurofascin in trans are important for neurite induction

Neurofascin is a member of the immunoglobulin superfamily involved in axon extension and fasciculation. Here we apply adenoviral short hairpin RNA (shRNA) expression in primary neurons, PC12–NIH/3T3 co‐cultures in combination with Luminex® assays, to demonstrate homophilic interactions of neurofascin for neurite outgrowth. An adenoviral vector was constructed for the expression of shRNA in primary tectal cells that inhibits gene expression similar to short interfering RNA. We demonstrate that after shRNA‐mediated knockdown neuronal neurofascin expression is important for neurite outgrowth on a neurofascin substrate. Neurite outgrowth assays reveal that neurite formation of PC12 cells is increased when neurofascin is overexpressed on both outgrowing PC12 cells and substrate NIH/3T3 cells, suggesting that neurofascin expression is also sufficient for neurite induction. Luminex technology for the analysis of protein–protein interactions showed homophilic binding of neurofascin to itself.

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra- and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.

GABAergic Synapses at the Axon Initial Segment of Basolateral Amygdala Projection Neurons Modulate Fear Extinction

Inhibitory synaptic transmission in the amygdala has a pivotal role in fear learning and its extinction. However, the local circuits formed by GABAergic inhibitory interneurons within the amygdala and their detailed function in shaping these behaviors are not well understood. Here we used lentiviral-mediated knockdown of the cell adhesion molecule neurofascin in the basolateral amygdala (BLA) to specifically remove inhibitory synapses at the axon initial segment (AIS) of BLA projection neurons. Quantitative analysis of GABAergic synapse markers and measurement of miniature inhibitory postsynaptic currents in BLA projection neurons after neurofascin knockdown ex vivo confirmed the loss of GABAergic input. We then studied the impact of this manipulation on anxiety-like behavior and auditory cued fear conditioning and its extinction as BLA related behavioral paradigms, as well as on long-term potentiation (LTP) in the ventral subiculum–BLA pathway in vivo. BLA knockdown of neurofascin impaired ventral subiculum–BLA–LTP. While this manipulation did not affect anxiety-like behavior and fear memory acquisition and consolidation, it specifically impaired extinction. Our findings indicate that modification of inhibitory synapses at the AIS of BLA projection neurons is sufficient to selectively impair extinction behavior. A better understanding of the role of distinct GABAergic synapses may provide novel and more specific targets for therapeutic interventions in extinction-based therapies.

Functional re-establishment of the perforant pathway in organotypic co-cultures on microelectrode arrays.

Co-cultures of entorhinal cortex (EC) and dentate gyrus (DG) explants are a useful model system to study the formation and stabilization of axonal projections. We adapted this model system to EC-DG co-cultures on microelectrode arrays (MEA) for the characterization of axonal projections on a functional level for days and weeks. EC and DG explants were placed on MEA to allow for the reconstitution of perforant pathway projections. Connections formed were characterized by morphological and electrophysiological analyses to verify characteristic features of perforant pathway signal transmission. Morphological analysis reveals proper projection of EC neurons into the molecular layer of the DG. Examination of synaptic transmission after high frequency stimulation imply unidirectional connections that used glutamate receptors of the AMPA/kainate type as main mediators of excitatory signal transmission. The system was evaluated by the introduction of the NCAM binding peptide C3d. In accordance with in vivo and in vitro experiments C3d modulated signal transmission by NCAM-related mechanisms resulting in morphological re-arrangements.