Nodes of Ranvier and axon initial segments are ankyrin G–dependent domains that assemble by distinct mechanismsAxon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and betaIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.
AAV vectors trigger DNA damage response-dependent pro-inflammatory signalling in human iPSC-derived CNS models and mouse brainAdeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless, dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS), taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated, the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition, transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together, our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Costa-Verdera et. al describe a mechanism by which AAV vector genomes activate P53-mediated signalling in CNS cells involving downstream STING activation and pro-inflammatory responses. Inhibition of either P53 or STING prevented target-cell apoptosis and inflammatory signalling.