Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

Pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) are considered to exert detrimental effects during brain trauma and in neurodegenerative disorders. Consistently, it has been demonstrated that IL-1β suppresses neurotrophin-mediated neuronal cell survival rendering neurons vulnerable to degeneration. Since neurotrophins are also well known to strongly influence axonal plasticity, we investigated here whether IL-1β has a similar negative impact on neurite growth. We analyzed neurite density and length of organotypic brain and spinal cord slice cultures under the influence of the neurotrophins NGF, BDNF, NT-3 and NT-4. In brain slices, only NT-3 significantly promoted neurite density and length. Surprisingly, a similar increase of neurite growth was induced by IL-1β. Additionally, both factors increased the number of brain slices displaying maximal neurite growth. Furthermore, the co-administration of IL-1β and NT-3 significantly increased the number of brain slices displaying maximal neurite growth compared to single treatments. These data indicate that these two factors synergistically stimulate two distinct aspects of neurite outgrowth, namely neurite density and neurite length from acute organotypic brain slices.

It is widely accepted that the angiotensin AT2-receptor (AT2R) has neuroprotective features. In the present study we tested pharmacological AT2R-stimulation as a therapeutic approach in a model of spinal cord compression injury (SCI) in mice using the novel non-peptide AT2R-agonist, Compound 21 (C21). Complementary experiments in primary neurons and organotypic cultures served to identify underlying mechanisms. Functional recovery and plasticity of corticospinal tract (CST) fibers following SCI were monitored after application of C21 (0.3mg/kg/dayi.p.) or vehicle for 4 weeks. Organotypic co-culture of GFP-positive entorhinal cortices with hippocampal target tissue served to evaluate the impact of C21 on reinnervation. Neuronal differentiation, apoptosis and expression of neurotrophins were investigated in primary murine astrocytes and neuronal cells. C21 significantly improved functional recovery after SCI compared to controls, and this significantly correlated with the increased number of CST fibers caudal to the lesion site. In vitro, C21 significantly promoted reinnervation in organotypic brain slice co-cultures (+50%) and neurite outgrowth of primary neurons (+25%). C21-induced neurite outgrowth was absent in neurons derived from AT2R-KO mice. In primary neurons, treatment with C21 further induced RNA expression of anti-apoptotic Bcl-2 (+75.7%), brain-derived neurotrophic factor (BDNF) (+53.7%), the neurotrophin receptors TrkA (+57.4%) and TrkB (+67.9%) and a marker for neurite growth, GAP43 (+103%), but not TrkC. Our data suggest that selective AT2R-stimulation improves functional recovery in experimental spinal cord injury through promotion of axonal plasticity and through neuroprotective and anti-apoptotic mechanisms. Thus, AT2R-stimulation may be considered for the development of a novel therapeutic approach for the treatment of spinal cord injury.

Precise crosstalk between the nervous and immune systems is important for neuroprotection and axon plasticity after injury. Recently, we demonstrated that IL-1β acts as a potent inducer of neurite outgrowth from organotypic brain slices in vitro, suggesting a potential function of IL-1β in axonal plasticity. Here, we have investigated the effects of IL-1β on axon plasticity during glial scar formation and on functional recovery in a mouse model of spinal cord compression injury (SCI). We used an IL-1β deficiency model (IL-1βKO mice) and administered recombinant IL-1β. In contrast to our hypothesis, the histological analysis revealed a significantly increased lesion width and a reduced number of corticospinal tract fibers caudal to the lesion center after local application of recombinant IL-1β. Consistently, the treatment significantly worsened the neurological outcome after SCI in mice compared with PBS controls. In contrast, the absence of IL-1β in IL-1βKO mice significantly improved recovery from SCI compared with wildtype mice. Histological analysis revealed a smaller lesion size, reduced lesion width and greatly decreased astrogliosis in the white matter, while the number of corticospinal tract fibers increased significantly 5 mm caudal to the lesion in IL-1βKO mice relative to controls. Our study for the first time characterizes the detrimental effects of IL-1β not only on lesion development (in terms of size and glia activation), but also on the plasticity of central nervous system axons after injury.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we use antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we use CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We identify Contactin-1 (Cntn1) as an AIS cell surface protein. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS extracellular matrix, and regulates AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.