AAV9-CMV-iCre

Neuronal remodeling and myelination are two fundamental processes during neural development. How they influence each other remains largely unknown, although their coordinated execution is critical for circuit function and is often disrupted in neuropsychiatric diseases. By modulating synaptic transmission, cytoskeletal dynamics, and axonal transport in mouse motor axons, researchers show that local axonal remodeling delays the onset of myelination and node formation. In contrast, glial differentiation does not dictate the outcome of axonal remodeling. Delayed myelination is not due to a limited supply of structural components of the axon-glial unit but rather to increased trafficking of signaling factors that initiate myelination, such as neuregulin. Furthermore, the transport of promyelinating signals is regulated by local cytoskeletal maturation associated with activity-dependent competition. These studies reveal an axon branch-specific fine-tuning mechanism that locally coordinates axonal remodeling and myelination.

To manipulate axonal microtubules, researchers genetically deleted the microtubule-severing enzyme spastin (spastin KO) and confirmed delayed axonal branch removal. Indeed, spastin loss led to accelerated node formation in competing axons (din) compared to WT. This represents a cell-autonomous effect in motor neurons, as confirmed by inducing a subset deletion in conditional spastin^fl/fl X TdTomato reporter mice using a Cre-encoding adeno-associated virus (AAV9-CMV-iCre; Figure 1). While axonal remodeling was again found to be delayed, node formation on competing branches was now accelerated (Figure 1C), where TdTomato expression indicated spastin loss. Overall, microtubule mass was increased in spastin-deleted terminal axon branches, while axon caliber was unaffected (Figure 1D), in stark contrast to the increased node formation on competing branches. Nrg1 type III is a candidate for a transported early myelinating signal because this signaling factor needs to reach a critical threshold locally to initiate axon-glial differentiation by activating downstream effectors in SCs, such as ERK1/2 and AKT. To investigate the function of Nrg1 type III during axon remodeling, researchers crossed floxed Nrg1 type III with TdTomato reporter mice and injected neonates with AAV9-CMV-iCre (Figure 1E). As expected, myelination was severely impaired in TdTomato-positive branches compared with internal control axons (Figure 1F-H).

Figure 1. AAV9-mediated spastin deletion promotes myelination on competing branches. (Wang M, et al., 2021)