Molecular mechanisms of neuronal connectivity

The differentiation of neuronal processes is essential for neuronal information processing. We are interested in proteins and microRNAs that regulate axonal branching, the elaboration of dendritic trees, the differentiation of dendritic spines and the formation of synapses.

Several years ago, we identified CALEB/NGC as a member of the EGF family of neural differentiation factors. We established a role of CALEB/NGC in regulating the complexity of dendritic trees and dendritic spines (Figure 1). Currently we are investigating the molecular mechanisms of how CALEB/NGC contributes to the differentiation of dendrites, spines, and synapses.

Figure 1. Primary hippocampal neurons in culture express either EGFP (left) or CALEB/NGC (right). CALEB/NGC increases the complexity of dendritic trees. Scholl analysis (red circles crossing dendrites) confirms that CALEB/NGC raises dendritic tree complexity by increasing dendritic branching. Scale bar, 20 mm. Modified from Brandt et al. (2007), EMBO J 26:2371-2386.

MicroRNAs are small noncoding RNAs that regulate gene expression. They are likely to have key roles in neuronal development and plasticity. We are interested in microRNA targets that contribute to the establishment of proper neuronal connectivity. Our focus is on the function of the small GTPase RhoG, the expression of which is regulated by the microRNA miR-124. This microRNA is specifically expressed in the nervous system. RhoG and miR-124-regulated control of RhoG expression play an important role in axonal and dendritic tree differentiation.

We address these topics with a wide spectrum of cell biological, biochemical and microscopic methods including primary neural cell culture, affinity chromatography, mass spectrometry, FRET, FRAP, immunohisto- and immunocytochemical techniques (Figure 2), in-utero-electroporation, and confocal microscopy combined with the analysis of knockout-mice.

Figure 2. Upper panel. Primary hippocampal neurons in culture were transfected with EGFP and stained for EGFP (GFP, to identify transfected neurons), the postsynaptic marker protein Shank2 (red) and the presynaptic marker protein Bassoon (blue). Bar, 20 mm.
Lower panel. A part of the dendritic tree (selected rectangles in upper panel) is shown in higher magnification. The GFP staining to the left highlights the dendrite and the dendritic spines, the combined GFP and Shank2 staining in the middle shows the postsynaptic compartments within the spines (yellow spots, arrows) and the triple staining of GFP, Shank2, and Bassoon to the right identifies synapses (matching presynaptic and postsynaptic compartments, white spots, arrows). Scale bar, 5 mm.

Molecular mechanisms of neuronal  progenitor cell development

Most of the neurons in the brain are derived from different types of neural progenitor cells (NPCs) as – for example – radial glial cells or intermediate neural progenitor cells. The main NPC types are able to perform different modes of cell division, e.g. symmetric proliferative, asymmetric proliferative, symmetric consumptive, or asymmetric consumptive divisions. For proper brain development to occur, a finely tuned balance of NPC proliferation versus differentiation, originating from an appropriate combination of the different cell division modes, is essential. We are analysing the molecular mechanisms of NPC development and showed that Polycystin-1 (PC1) and Polycystin-2 (PC2), two proteins heavily involved in the pathology of polycystic kidney disease, regulate the decision of NPCs to proliferate for expansion, or for self-renewal and differentiation to generate neurons (Figure 3).

Figure 3. Reduced expression of PC1 or PC2 expression promotes symmetric divisions of NPCs. Neocortical NPCs were co-transfected with the constructs indicated to the left, subjected to 6 h of BrdU labeling one day after transfection, and fixed 18 h after the BrdU labeling. The GFP signal is derived from a sensor construct monitoring active Notch signaling. In the control (upper panel), a cell pair is shown (marked by an oval window) with asymmetric distribution of Notch activity following cell division. Knockdown of either PC1 or PC2 leads to an increase in the number of cell pairs with symmetric distribution of Notch signaling after cell division (middle and lower panels). Scale bar, 15 mm. Adopted from Winokurow and Schumacher (2019), Cell Mol Life Sci 76:2851-2869.

Project Team

Univ.-Prof. Dr. rer. physiol. Stefan Schumacher (group leader)
Dr. Natalie Winokurow (Scientist)
Anne Lehner (Technical assistant)
Alexandra Liebaug (Technical assistant)

Link to our Publications