Hyukmin Kim

Modality-Based Organization of Ascending Somatosensory Axons in the Direct Dorsal Column Pathway

The long-standing doctrine regarding the functional organization of the direct dorsal column (DDC) pathway is the “somatotopic map” model, which suggests that somatosensory afferents are primarily organized by receptive field instead of modality. Using modality-specific genetic tracing, here we show that ascending mechanosensory and proprioceptive axons, two main types of the DDC afferents, are largely segregated into a medial–lateral pattern in the mouse dorsal column and medulla. In addition, we found that this modality-based organization is likely to be conserved in other mammalian species, including human. Furthermore, we identified key morphological differences between these two types of afferents, which explains how modality segregation is formed and why a rough “somatotopic map” was previously detected. Collectively, our results establish a new functional organization model for the mammalian direct dorsal column pathway and provide insight into how somatotopic and modality-based organization coexist in the central somatosensory pathway.

Sensory Axon Regeneration: A Review from an in vivo Imaging Perspective.

Injured primary sensory axons fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Why axons stop or turn around at the DREZ has generally been attributed to growth-repellent molecules associated with astrocytes and oligodendrocytes/myelin. The available evidence challenges the contention that these inhibitory molecules are the critical determinant of regeneration failure. Recent imaging studies that directly monitored axons arriving at the DREZ in living animals raise the intriguing possibility that axons stop primarily because they are stabilized by forming presynaptic terminals on non-neuronal cells that are neither astrocytes nor oligodendrocytes. These observations revitalized the idea raised many years ago but virtually forgotten, that axons stop by forming synapses at the DREZ.

NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons.

YAP/TAZ initiate and maintain Schwann cell myelination

Nuclear exclusion of the transcriptional regulators and potent oncoproteins, YAP/TAZ, is considered necessary for adult tissue homeostasis. Here we show that nuclear YAP/TAZ are essential regulators of peripheral nerve development and myelin maintenance. To proliferate, developing Schwann cells (SCs) require YAP/TAZ to enter S-phase and, without them, fail to generate sufficient SCs for timely axon sorting. To differentiate, SCs require YAP/TAZ to upregulate Krox20 and, without them, completely fail to myelinate, resulting in severe peripheral neuropathy. Remarkably, in adulthood, nuclear YAP/TAZ are selectively expressed by myelinating SCs, and conditional ablation results in severe peripheral demyelination and mouse death. YAP/TAZ regulate both developmental and adult myelination by driving TEAD1 to activate Krox20. Therefore, YAP/TAZ are crucial for SCs to myelinate developing nerve and to maintain myelinated nerve in adulthood. Our study also provides a new insight into the role of nuclear YAP/TAZ in homeostatic maintenance of an adult tissue. DOI: http://dx.doi.org/10.7554/eLife.20982.001

Nanotherapeutics of PTEN Inhibitor with Mesoporous Silica Nanocarrier Effective for Axonal Outgrowth of Adult Neurons

Development of therapeutic strategies such as effective drug delivery is an urgent and yet unmet need for repair of damaged nervous systems. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulates axonal regrowth of central and peripheral nervous systems; its inhibition, meanwhile, facilitates axonal outgrowth of injured neurons. Here we show that nanotherapeutics based on mesoporous silica nanoparticles loading PTEN-inhibitor bisperoxovanadium (BpV) are effective for delivery of drug molecules and consequent improvement of axonal outgrowth. Mesoporous nanocarriers loaded BpV drug at large amount (27 μg per 1 mg of carrier), and released sustainably over 10 d. Nanocarrier-BpV treatment of primary neurons from the dorsal root ganglions (DRGs) of rats and mice at various concentrations induced them to actively take up the nanocomplexes with an uptake efficiency as high as 85%. The nanocomplex-administered neurons exhibited significantly enhanced axonal outgrowth compared with those treated with free-BpV drug. The expression of a series of proteins involved in PTEN inhibition and downstream signaling was substantially up-/down-regulated by the nanocarrier-BpV system. Injection of the nanocarriers into neural tissues (DRG, brain cortex, and spinal cord), moreover, demonstrated successful integration into neurons, glial cells, oligodendrocytes, and macrophages, suggesting the possible nanotherapeutics applications in vivo. Together, PTEN-inhibitor delivery via mesoporous nanocarriers can be considered a promising strategy for stimulating axonal regeneration in central and peripheral nervous systems.

Post-injury induction of activated ErbB2 selectively hyperactivates denervated Schwann cells and promotes robust dorsal root axon regeneration

Following nerve injury, denervated Schwann cells (SCs) convert to repair SCs, which enable regeneration of peripheral axons. However, the repair capacity of SCs and the regenerative capacity of peripheral axons are limited. In the present studies we examined a potential therapeutic strategy to enhance the repair capacity of SCs, and tested its efficacy in enhancing regeneration of dorsal root (DR) axons, whose regenerative capacity is particularly weak. We used male and female mice of a doxycycline-inducible transgenic line to induce expression of constitutively active ErbB2 (caErbB2) selectively in SCs after DR crush or transection. Two weeks after injury, injured DRs of induced animals contained far more SCs and SC processes. These SCs had not redifferentiated and continued to proliferate. Injured DRs of induced animals also contained far more axons that regrew along SC processes past the transection or crush site. Remarkably, SCs and axons in uninjured DRs remained quiescent, indicating that caErbB2 enhanced regeneration of injured DRs, without aberrantly activating SCs and axons in intact nerves. We also found that intraspinally expressed glial cell line-derived neurotrophic factor (GDNF), but not the removal of chondroitin sulfate proteoglycans, greatly enhanced the intraspinal migration of caErbB2-expressing SCs, enabling robust penetration of DR axons into the spinal cord. These findings indicate that SC-selective, post-injury activation of ErbB2 provides a novel strategy to powerfully enhance the repair capacity of SCs and axon regeneration, without substantial off-target damage. They also highlight that promoting directed migration of caErbB2-expressing SCs by GDNF might be useful to enable axon regrowth in a non-permissive environment. SIGNIFICANCE STATEMENT Repair of injured peripheral nerves remains a critical clinical problem. We currently lack a therapy that potently enhances axon regeneration in patients with traumatic nerve injury. It is extremely challenging to substantially increase the regenerative capacity of damaged nerves without deleterious off-target effects. It was therefore of great interest to discover that caErbB2 markedly enhances regeneration of damaged dorsal roots, while evoking little change in intact roots. To our knowledge, these findings are the first demonstration that repair capacity of denervated SCs can be efficaciously enhanced without altering innervated SCs. Our study also demonstrates that oncogenic ErbB2 signaling can be activated in SCs but not impede transdifferentiation of denervated SCs to regeneration-promoting repair SCs.

PTEN negatively regulates the cell lineage progression from NG2+ glial progenitor to oligodendrocyte via mTOR-independent signaling

Oligodendrocytes (OLs), the myelin-forming CNS glia, are highly vulnerable to cellular stresses, and a severe myelin loss underlies numerous CNS disorders. Expedited OL regeneration may prevent further axonal damage and facilitate functional CNS repair. Although adult OL progenitors (OPCs) are the primary players for OL regeneration, targetable OPC-specific intracellular signaling mechanisms for facilitated OL regeneration remain elusive. Here, we report that OPC-targeted PTEN inactivation in the mouse, in contrast to OL-specific manipulations, markedly promotes OL differentiation and regeneration in the mature CNS. Unexpectedly, an additional deletion of mTOR did not reverse the enhanced OL development from PTEN-deficient OPCs. Instead, ablation of GSK3β, another downstream signaling molecule that is negatively regulated by PTEN-Akt, enhanced OL development. Our results suggest that PTEN persistently suppresses OL development in an mTOR-independent manner, and at least in part, via controlling GSK3β activity. OPC-targeted PTEN-GSK3β inactivation may benefit facilitated OL regeneration and myelin repair.

Time-Lapse In Vivo Imaging of Dorsal Root Nerve Regeneration in Mice

Primary sensory axon injury is common after spinal cord and root injuries and causes patients to suffer chronic pain and persistent loss of sensation and motor coordination. The devastating consequences of such injuries are due primarily to the failure of severed axons to regenerate within the damaged CNS. Our understanding of the molecular and cellular events that play key roles in preventing or promoting functional regeneration is far from complete, in part because complex and dynamic changes associated with nerve injury have had to be deduced from comparisons of static images obtained from multiple animals after their death. Revolutionary innovations in optics and mouse transgenics now permit real-time monitoring of regenerating dorsal root axons directly in living animals. Here, we describe detailed procedures for repetitive monitoring of identified axons in a lumbar dorsal root over hours to weeks using both widefield and two-photon microscopes. We also discuss the strengths and limitations of in vivo imaging and provide suggestions based on our own experience for troubleshooting issues associated with repeated anesthetization, an extensive laminectomy, and post-op care. These techniques provide the unprecedented opportunity to obtain novel insights into why sensory axons fail to reenter the spinal cord.

Sensory Nerve Regeneration at the CNS-PNS Interface

Over a century ago, Ramon y Cajal, using the Golgi staining technique to label a subset of dorsal root ganglion (DRG) axons, showed that injured DR axons regenerate within the root but fail to re-enter the adult spinal cord. As shown in his drawing (Fig. 1), DR axons grow away from (arrow), or stop at (arrowheads), the junction between the CNS and PNS, termed the dorsal root entry zone (DREZ). Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the transitional zone between the CNS and PNS. Why regeneration fails at DREZ has remained an interesting issue both because dorsal root injuries are common and because DREZ serves as an excellent model system for studying the reasons for the failure of CNS regeneration.