Neurons in the central nervous system (CNS) fail to regenerate axons after injuries due to the diminished intrinsic axon growth capacity of mature neurons and the hostile extrinsic environment composed of a milieu of inhibitory factors. Recent studies revealed that targeting a particular group of extracellular inhibitory factors is insufficient to trigger long-distance axon regeneration. Instead of antagonizing the growing list of impediments, tackling a common target that mediates axon growth inhibition offers an alternative strategy to promote axon regeneration. Neuronal growth cone, the machinery that derives axon extension, is the final converging target of most, if not all, growth impediments in the CNS. In this study, we aim to promote axon growth by directly targeting the growth cone. Here we report that pharmacological inhibition or genetic silencing of nonmuscle myosin II (NMII) markedly accelerates axon growth over permissive and nonpermissive substrates, including major CNS inhibitors such as chondroitin sulfate proteoglycans and myelin-associated inhibitors. We find that NMII inhibition leads to the reorganization of both actin and microtubules (MTs) in the growth cone, resulting in MT reorganization that allows rapid axon extension over inhibitory substrates. In addition to enhancing axon extension, we show that local blockade of NMII activity in axons is sufficient to trigger axons to grow across the permissive-inhibitory border. Together, our study proposes NMII and growth cone cytoskeletal components as effective targets for promoting axon regeneration.myelin | glial scar | multi-compartment neuronal culture chamber

We describe a novel valve-based microfluidic axon injury micro-compression (AIM) platform that enables focal and graded compression of micron-scale segments of single central nervous system (CNS) axons. The device utilizes independently controlled "push-down" injury pads that descend upon pressure application and contact underlying axonal processes. Regulated compressed gas is input into the AIM system and pressure levels are modulated to specify the level of injury. Finite element modeling (FEM) is used to quantitatively characterize device performance and parameterize the extent of axonal injury by estimating the forces applied between the injury pad and glass substrate. In doing so, injuries are normalized across experiments to overcome small variations in device geometry. The AIM platform permits, for the first time, observation of axon deformation prior to, during, and immediately after focal mechanical injury. Single axons acutely compressed (~5 s) under varying compressive loads (0-250 kPa) were observed through phase time-lapse microscopy for up to 12 h post injury. Under mild injury conditions (< 55 kPa) ~73% of axons continued to grow, while at moderate (55-95 kPa) levels of injury, the number of growing axons dramatically reduced to 8%. At severe levels of injury (> 95 kPa), virtually all axons were instantaneously transected and nearly half (~46%) of these axons were able to regrow within the imaging period in the absence of exogenous stimulating factors.

Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons.The interactions of the human neurotrophic herpesvirus varicella-zoster virus (VZV) with neurons have proven difficult to study because the virus shows fairly strict human specificity, and small-animal models do not fully recapitulate human disease. In humans, primary VZV infection follows viral inhalation and subsequent systemic delivery to the deep dermis of the skin via hemopoietic cells. In the course of the resulting disease (chickenpox), VZV infects sensory and sympathetic ganglion neurons, where it establishes a long period of latency. The infection of neurons may take place in the ganglia by circulating VZV-infected lymphocytes, or by virus infecting cutaneous nerve endings being retrogradely transported in the axon to the neuronal somata, as is the case with herpes simplex virus (HSV). VZV reactivation often leads to herpes zoster (shingles), a disease that is frequently associated with severe, debilitating, and often long-lasting intractable pain (postherpetic neuralgia) that is more often than not refractory to therapy.Few model systems of neuronal VZV infection have been developed. Two in vitro models are VZV infection of dissociated human neurons and intact human fetal dorsal root ganglia (DRG) (8, 9, 10). These studies have shed some light on VZV-neuronal interactions, demonstrating, for example, that VZV exerts antiapoptotic activities in neurons in the short term (maximum, 5 days) and that, unlike infected fibroblasts, infectious VZV is released from neurons.A human fetal DRG-SCID mouse model (22, 29; reviewed in reference 30) has al...