Transneuronal Delivery of Cytokines to Stimulate Mammalian Spinal Cord Regeneration.

The spinal cord contains multiple fiber tracts necessary for locomotion. However, as a part of the central nervous system, they are extremely limited in regenerating after injury. Many of these key fiber tracts originate from deep brain stem nuclei that are difficult to access. Here we detail a new methodology that achieves functional regeneration in mice after a complete spinal cord crush, describing the crushing procedure itself, intracortical treatment application, and a set of appropriate validation steps. The regeneration is achieved by the one-time transduction of neurons in the motor cortex with a viral vector expressing the designer cytokine hIL-6. This potent stimulator of the JAK/STAT3 pathway and regeneration is transported in axons and then transneuronally delivered to critical deep brain stem nuclei via collateral axon terminals, resulting in previously paralyzed mice walking again after 3-6 weeks. With no previously known strategy accomplishing this degree of recovery, this model is well suited to studying the functional impact of compounds/treatments currently only known to promote anatomical regeneration.

Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development.

It is highly desirable to specify human developmental principles in an appropriate human model with advanced genetic tools. However, genetically engineering human cells with lineage-tracing systems has not been achieved. Here we introduce strategies to construct lineage-tracing systems in human embryonic stem cells (hESCs). The AAVS1 locus was suitable for the integration of the conditional reporter. The Cre-LoxP and Flp-FRT systems were highly sensitive, which may cause inaccurate lineage labeling in human cells. The recombination sensitivity and tracing fidelity could be finely tuned by modification of the LoxP recombination site. Moreover, tamoxifen-controllable CreERT2-LoxP and FlpERT2-FRT systems showed compelling advantages in tightly tracing human lineages temporally. In proof-of-principle experiments, we traced human PAX6+ neuroectoderm cells and revealed their full neural lineage differentiation potency both in vitro and in vivo. Devising and optimizing of lineage-tracing systems in hESCs will thus set up a solid foundation for human developmental studies.

CXCR4/SDF1 signalling promotes sensory neuron clustering in vitro.

During the development of the peripheral nervous system, a subgroup of neural crest cells migrate away from the neural tube and coalesce into clusters of sensory neurons (ganglia). Mechanisms involved in the formation of the dorsal root ganglia (DRG) from neural crest cells are currently unclear. Mice carrying mutations in Cxcr4, which is known to control neural crest migration, exhibit malformed DRG. In order to investigate this phenomenon, we modelled sensory neuron differentiation in vitro by directing the differentiation of human induced pluripotent stem cells into sensory neurons under SDF1 (agonist), AMD3100 (antagonist) or control conditions. There we could show a marked effect on the clustering activity of the neurons in vitro, suggesting that CXCR4 signalling is involved in facilitating DRG condensation.

VEGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone.

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott-Aldrich-Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP.

Smad5 acts as an intracellular pH messenger and maintains bioenergetic homeostasis.

Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.