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Ciliary neurotrophic factor (Cntf) acts as a differentiation and survival factor for different types of neurons and glial cells. It is expressed by peripheral Schwann cells and astrocytes in the central nervous system and mediates its effects via a receptor complex involving CntfRα, LifRß and gp130, leading to downstream activation of Stat3. Recent studies by our group have shown that Cntf modulates neuronal microtubule dynamics via Stat3/stathmin interaction. In a mouse model for motor neuron disease, i.e. pmn, Cntf is able to rescue axonal degeneration through Stat3/stathmin signaling. While these findings suggest a role of Cntf in controlling axonal functions in the neuromuscular system, additional data indicate that Cntf might also play a role in synaptic plasticity in the hippocampus. Electrophysiological recordings in hippocampal organotypic cultures and acute slices revealed a deficit in long-term potentiation (LTP) in Cntf -/- mice. This deficit was rescued by 24 h stimulation with Cntf, combined with an acute application of Cntf during LTP-measurements indicating that Cntf is both necessary and sufficient for hippocampal LTP, and possibly synaptic plasticity. Therefore, Cntf knockout mice were investigated to elucidate this possible role of Cntf in hippocampal LTP and synaptic plasticity.
First, we validated the presence of Cntf in the target tissue: in the hippocampus, Cntf was localized in Gfap-positive astrocytes surrounding small blood vessels in the fissure and in meningeal areas close to the dentate gyrus. Laser micro-dissection and qPCR analysis showed a similar distribution of Cntf-coding mRNA validating the obtained immunofluorescent results. Despite the strong LTP deficit in organotypic cultures, in vivo behavior of Cntf -/- mice regarding hippocampus-dependent learning and anxiety-related paradigms was largely inconspicuous. However, western blot analysis of hippocampal organotypic cultures revealed a significant reduction of pStat3 levels in Cntf -/- cultures under baseline conditions, which in turn were elevated upon Cntf stimulation. In order to resolve and examine synaptic structures we turned to in vitro analysis of cultured hippocampal neurons which indicated that pStat3 is predominantly located in the presynapse. In line with these findings, presynapses of Cntf -/- cultures were reduced in size and when in contact to astrocytes, contained less pStat3 immunoreactivity compared to presynapses in wildtype cultures.
In conclusion, our findings hypothesize that despite of a largely inconspicuous behavioral phenotype of Cntf -/- mice, Cntf appears to have an influence on pStat3 levels at hippocampal synapses. In a next step these two key questions need to be addressed experimentally: 1) is there a compensatory mechanism by members of the Cntf family, possibly downstream of pStat3, which explains the in vivo behavioral results of Cntf -/- mice and can likewise account for the largely inconspicuous phenotype in CNTF-deficient humans? 2) How exactly does Cntf influence LTP through Stat3 signaling? To unravel the underlying mechanism further experiments should therefore investigate whether microtubule dynamics downstream of Stat3 and stathmin signaling are involved in the Cntf-induced modulation of hippocampal synaptic plasticity, similar to as it was shown in motoneurons.
A mouse model for genetic deletion of presynaptic BDNF from adult hippocampal mossy fiber terminals
(2020)
Brain-derived neurotrophic factor (BDNF) is a modulator and mediator of structural and functional plasticity at synapses in the central nervous system. Despite our profound knowledge about the synaptic function of BDNF at synapses, it is still controversially discussed whether synaptic BDNF acts primarily from pre- or postsynaptic sites. In the central nervous system, several studies show that mossy fiber (MF) projections formed by hippocampal granule neurons store the highest amount of BDNF. However, immunofluorescence and RNA labelling studies suggest that MF BDNF is primarily produced by granule neurons. Multiple other studies prefer the view that BDNF is primarily produced by postsynaptic neurons such as CA3 pyramidal neurons. Here, we question whether the BDNF, which is stored in the mossy fiber synapse, is primarily produced by granule neurons or whether by other cells in the MF-CA3 microcircuit. After standardization of immunolabelling of BDNF, confocal imaging confirmed the localization of BDNF in presynaptic MF terminals. This anterograde location of synaptic BDNF was also found in distinct regions of the fear and anxiety circuit, namely in the oval nucleus of the bed nucleus stria terminals (ovBNST) and in the central amygdala. To find out whether the presynaptic BDNF location is due to protein translation in the corresponding presynaptic dentate gyrus (DG) granule neuron, we developed and characterized a mouse model that exhibits BDNF deletion specifically from adult DG granule neurons. In this mouse model, loss of presynaptic BDNF immunoreactivity correlated with the specific Creactivity in granule neurons, thus confirming that MF BDNF is principally released by granule neurons. After BDNF deletion from granule neurons, we observed more immature neurons with widely arborized dendritic trees. This indicated that local BDNF deletion also affects the local adult neurogenesis, albeit Cre-mediated BDNF deletion only occur in adult granule neurons. Since BDNF is a master regulator of structural synaptic plasticity, it was questioned whether it is possible to visualize presynaptic, synapse-specific, structural plasticity in mossy fiber synapses. It was established that a combination of Cre-techniques together with targeting of GFP to membranes with the help of palmitoylation / myristoylation anchors was able to distinctly outline the synaptic structure of the BDNF-containing MF synapse. In summary, the mouse model characterized in here is suited to investigate the synaptic signalling function of presynaptic BDNF at the mossy fiber terminal, a model synapse to investigate microcircuit information processing from molecule to behaviour.