Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules.
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Acknowledgments
We are grateful to A. McKinney and S. Hugel for help with imaging and to J. M. Fritschy, P. Scheiffele and J. P. Loeffler for kindly providing us antibodies. We thank L. Dimou for help with animal breeding, F. Christ for excellent technical support and R. Schöb and E. Hochreutener for help with the figures. This study was supported by grants of the Swiss National Science Foundation (31-63633.00), the NCCR “Neural Plasticity and Repair” of the Swiss National Science Foundation, the Spinal Cord Consortium of the Christopher Reeve Foundation (Springfield, NJ, USA), the DFG-SNF Transregio Sonderforschungsbereich Konstanz-Zurich and the EU-NeuroNetwork Project.
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Fig9
Specificity of the rabbit antiserum “Laura” (173b) antibody for Nogo-A. Immunoblotting of PC12 cells with crude antiserum (AS) and IgG purified Laura antibody shows a single band corresponding to Nogo-A. Note the specificity of the antibody and the absence of unspecific bands (JPG 95 kb)
Fig10
Nogo-A deletion does not affect the maintenance of the Purkinje cell – Deep Cerebellar Nuclei synapse. A: The mean density (± SEM) of overall Synaptophysin staining of DCN sections is similar in Nogo-A knockout and wildtype control mice. B: The number of GAD-65 positive Purkinje boutons is not significantly changed in mice lacking Nogo-A. C-F: Threshold confocal pictures of Purkinje boutons labeled for GAD-65 from wildtype (C-D) and Nogo-A knockout (E-F) mice were used for quantification of the number of Purkinje cell boutons per 100µm of DCN cell body membrane. Scale bar: 10µm (JPG 704 kb)
Fig11
Worsening of ataxia / clasping response in 14 months old L7-Nogo-A transgenic mice. Nogo-A overexpression in Purkinje neurons induces a progressive reflex reversal resulting in limb flexion instead of extension (clasping reflex) in the tail suspension test. A: at 14 months the failure of limb extension was even more severe than at 10 months (** p < 0.01, *** p < 0.001). B: correlation between Nogo-A protein expression level shown by immunofluoresence and clasping score in individual mice demonstrates that the defects displayed in the clasping reflex are positively correlated to the level of Nogo-A protein expression (** p < 0.01, *** p < 0.001) (JPG 158 kb)
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Aloy, E.M., Weinmann, O., Pot, C. et al. Synaptic destabilization by neuronal Nogo-A. Brain Cell Bio 35, 137–157 (2006). https://doi.org/10.1007/s11068-007-9014-3
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DOI: https://doi.org/10.1007/s11068-007-9014-3